Streptococcus mutans is a bacterium that causes oral diseases. Plaque, a biofilm produced by S. mutans and other bacteria, makes it difficult to remove cariogenic oral microorganisms, including biofilm producers. Glucan synthesis by glucosyltransferase is one of the mechanisms underlying plaque formation. This study demonstrates the effectiveness of inhibiting biofilm formation by interfering with the glucosyltransferase activity of S. mutans using edible herbal medicines.S. mutans. Enzyme kinetic analysis identified the mechanism by which glycyrrhizin and ellagic acid inhibit enzyme activity.This study investigated the inhibitory activity of Glycyrrhizae Radix extract, Rubi Fructus extract, glycyrrhizin from Glycyrrhizae Radix, and ellagic acid from Rubi Fructus against glucosyltransferase activity of The conditions for synergistically inhibiting biofilm formation by combining Glycyrrhizae Radix and Rubi Fructus extracts were identified. Biofilm formation was also synergistically inhibited by mixing their respective active constituents, glycyrrhizin and ellagic acid. Glycyrrhizin and ellagic acid inhibited glucosyltransferase via noncompetitive and uncompetitive mechanisms, respectively, indicating that they inhibit it via distinct mechanisms.S. mutans.This study presents an effective oral hygiene method using the synergistic activity of two natural plant extracts to inhibit biofilm formation through different inhibitory mechanisms against glucosyltransferase of The online version contains supplementary material available at 10.1186/s12906-023-03861-9. Streptococcus mutans is a major cause of dental caries and a representative bacterium that forms biofilm in the oral cavity, which is called plaque [S. mutans glucosyltransferase (GTase) synthesizes sticky glucan from sucrose to promote the formation of dental biofilm and the attachment of oral microorganisms. Oral microorganisms, including S. mutans, produce organic acids from carbohydrates and accelerate the progression of dental caries [Dental caries is an infectious oral disease that causes progressive destruction and loss of tooth enamel . Streptod plaque . Furtherl caries . TherefoS. mutans adhere to the surface of teeth [GTase synthesizes water-soluble and/or water-insoluble glucan. Exopolysaccharide-based insoluble glucan is a significant component in the formation of dental biofilm  and helpof teeth –7. Becauof teeth , 9, alteof teeth , 11, andof teeth  can subsof teeth , 14. Inhof teeth , 16.S. mutans biofilm formation. In addition, it is necessary to identify the active constituents of plant extracts that can be used in combination and their biological mechanisms of action.In recent decades, chlorhexidine and sodium fluoride have been used to prevent oral diseases; however, they are irritative and have negative consequences, such as the emergence of resistant strains –19. AlteS. mutans biofilm formation via different mechanisms [S. mutans [Rubus coreanus) that inhibits GTase activity but not the growth of S. mutans [Our previous studies recently proposed two materials that strongly inhibit chanisms , 29. One. mutans .Glycyrrhizae Radix and Rubi Fructus are sweet, medicinal, or functional foods with a long history of use. In numerous studies, their biological activities and active constituents have also been suggested. Glycyrrhizae Radix is recognized for its anti-inflammatory , antioxiS. mutans by combining the extracts of Glycyrrhizae Radix and Rubi Fructus is proposed. To this end, this study proposes an extraction method for Rubi Fructus in addition to the extraction method for Glycyrrhizae Radix from the previous study [S. mutans in the two proposed extracts.In this study, a method for inhibiting the biofilm formation of us study , as wellStreptococcus mutans GS-5, a serotype c strain, was provided by LG Household & Health Care Ltd. [S. mutans stored in 25% (v/v) glycerol stock at − 80 °C was streaked on a BHI agar plate and incubated at 37 °C for 2 days. The BHI medium was inoculated with a single colony from the BHI agar plates and incubated at 37 °C for 24 h.are Ltd. . Brain hGlycorrhizin  and ellagic acid dihydrate  were purchased from Tokyo Chemical Industry Co., Ltd.  and used as standard compounds for the analyses of Glycyrrhizae Radix and Rubi Fructus, respectively. Glycyrrhizin (100 mM) and ellagic acid (5 mM) were dissolved in dimethyl sulfoxide (DMSO) before use.Dried Glycyrrhizae Radix, purchased from Jiwoondang Herbal Medicine Store , was ground to a powder of ≤1 mm. Then, the extract solution was mixed at 8 mL per 1 g of Glycyrrhizae Radix powder and incubated at 83 °C for 3 h. The solid powder was eliminated by filtration using a 75 μm–pore cartridge filter. The solvent was mixed again at 2 mL per 1 g of Glycyrrhizae Radix powder and incubated at 83 °C for 2 h. The residual powder was removed once more with the use of the same cartridge filter. The powdered mixture of the filtered primary and secondary extracts was produced via spray drying.Dried Rubi Fructus from Jiwoondang Herbal Medicine Store  was ground to a powder with a particle size of ≤1 mm. Next, the solvent was mixed at 10 mL per 1 g of the Rubi Fructus powder and incubated at 100 °C (water) or 70 °C (50 and 90% ethanol) for 3 h. Following filtration with a cartridge filter, the extract was spray-dried into a powder. The solid yields were 12.7% with water, 21.2% with 50% ethanol, and 10.7% with 90% ethanol.Glycyrrhizae Radix and Rubi Fructus were formally identified by the Herbal Medicine Store, where they were purchased. All extract powders were vacuum-sealed and stored at − 80 °C until use. Before use, extract powders of Glycyrrhizae Radix and Rubi Fructus were dissolved in distilled water.S. mutans, with an absorbance of 0.05 at 600 nm, was inoculated into each well. After 24 h of incubation at 37 °C, the absorbance at 595 nm was measured to determine cell growth using an Opsys MR microplate reader . The planktonic cells were then washed with distilled water. The biofilm was stained with 1% crystal violet and then rinsed with distilled water. For the quantitative analysis of biofilm formation, crystal violet remaining in the biofilm was eluted with 95% ethanol, and the absorbance at 595 nm was measured. The relative amount of biofilm was calculated by comparison with the control value.Microplate biofilm formation was measured using crystal violet staining method . In ordeS. mutans which was stored at − 80 °C, was inoculated on a BHI agar plate and cultured at 37 °C for 2 days. A single colony was inoculated into 40 mL of BHI medium and incubated at 37 °C for 24 h. The preculture cells were inoculated onto 1 L of BHI medium to reach Abs600 = 0.05 and incubated at 37 °C for 24 h. The cells were separated from the supernatant using a centrifuge  at 1500×g and 4 °C for 10 min. This cell-free supernatant was combined with cold 95% ethanol and incubated at 4 °C for 24 h. The supernatant was discarded after centrifugation at 1500×g and 4 °C for 10 min to obtain precipitated GTase. Precipitated GTase was washed with 10 mL of 60 mM potassium phosphate buffer (pH 6.8). The GTase was homogenized with an additional 10 mL of 60 mM potassium phosphate buffer (pH 6.8) and stored at − 80 °C.Glucosyltransferase activity was measured using a modified version of the method described in previous reports , 44. S. 3. For the reaction, 800 μL of the substrate solution, 50 μL of GTase, 50 μL of the testing sample, and 100 μL of distilled water were mixed and incubated at 37 °C for 24 h. After incubation, insoluble glucan was homogenized for 5 s using a Sonic Dismembrator  with the power of scale 4. After centrifugation at 1500×g and 4 °C for 10 min, most of the supernatant was removed, leaving ~ 200 μL. The pelleted insoluble glucan was homogenized using sonication under the same conditions mentioned above. After transferring 200 μL into a 96-well polystyrene microplate , absorbance at 540 nm was measured using an Opsys MR™ microplate reader. For the control experiments, distilled water was substituted for the test sample. To calculate the relative GTase activity, the sample value was compared to the control value.By analyzing the amount of insoluble glucan produced from sucrose during the enzymatic reaction, the GTase activity was determined. The substrate for the reaction was 1.25% (w/v) sucrose in a 60 mM potassium phosphate buffer at pH 6.8 with 0.025% NaNThe concentration of ellagic acid in the Rubi Fructus extract was determined using high-performance liquid chromatography (HPLC) in accordance with the method described in a previous report . Each spUsing a 96-well microplate, the synergistic activity of extract combinations was evaluated . The relKm) and maximum reaction rate (Vmax) of GTase were determined using a slightly modified version of a previously described method [Vmax and Km were calculated using the Lineweaver–Burk equation. To determine the type of inhibition, the rate of change of the reaction upon the addition of glycyrrhizin or ellagic acid was compared.Michaelis–Menten constants  was utilized . The results were determined using the S. mutans include 1) inhibiting the growth of S. mutans, 2) inhibiting the biofilm formation of S. mutans (including inhibiting glucan synthesis by GTase), and 3) employing alternative sweeteners that cannot be exploited by S. mutans [Methods for preventing oral diseases caused by . mutans .R. ulmifolius was found to inhibit the biofilm formation of Staphylococcus aureus [R. idaeus extract inhibited the biofilm formation of Candida albicans without antifungal activity [Pseudomonas libanensis, Serratia marcescens, Acinetobacter sp., Bacillus amyloliquefaciens, Hafnia paralvei, Obesumbacterium proteus, and Pseudomonas aeruginosa [Rubus spp. extract against microbial biofilm is strain-specific.In a previous study, ellagic acid rhamnoside from s aureus , and theactivity . Howeverruginosa . These fS. mutans [S. mutans biofilm formation  and maximum velocity (Vmax) of GTase isolated from S. mutans, the enzymatic activity was measured using various concentrations of sucrose, and the Lineweaver–Burk equation was used to plot the obtained results (Fig. Km and Vmax values of GTase were 0.99 mM and 0.218 Abs540/mL/30 min, respectively. The Km value for glycyrrhizin was 0.93 mM, which was comparable to that of the control GTase. However, the Km value for ellagic acid was 0.57 mM, which was 42% lower than that of the control GTase. Vmax values for glycyrrhizin and ellagic acid were 0135 and 0.105 Abs540/mL/30 min, representing decreases of 38 and 52%, respectively. These results indicated that glycyrrhizin is a noncompetitive inhibitor and ellagic acid is an uncompetitive inhibitor of GTase, indicating that these two compounds inhibit the enzyme in different ways.To determine the Michaelis–Menten kinetic constant (lts Fig. . WithoutS. mutans [S. mutans through mixed-type inhibition [S. mutans biofilm formation was inhibited synergistically by extracts of Glycyrrhizae Radix and Rubi Fructus containing these two different inhibitors as the main active substances, respectively.In previous studies, inhibitors of GTase activity with diverse inhibitory mechanisms were described. For instance, polyphenols of oolong tea noncompetitively inhibited the glucan-binding domain of the GTase of . mutans . Meanwhihibition . Furtherhibition . Accordihibition . In thisS. mutans’ formation of dental biofilm. Rubi Fructus water extract inhibited GTase activity and biofilm formation without inhibiting the growth of S. mutans. Glycyrrhizin and ellagic acid were found to be noncompetitive and uncompetitive inhibitors of S. mutans GTase, respectively. The combination of glycyrrhizin of Glycyrrhizae Radix and ellagic acid of Rubi Fructus synergistically inhibited biofilm formation, and the combination of Rubi Fructus extract with Glycyrrhizae Radix extract exerting different mechanisms of action showed a strong ability to inhibit biofilm formation at low concentrations. Thus, it is possible to develop effective products for preventing oral diseases caused by S. mutans dental biofilm using the proposed combination’s synergistic activity.This study revealed the synergistic activity of a combination of two different natural compounds as a potential method for inhibiting Additional file 1: Supplementary Fig. 1. Effect of Glycyrrhizae Radix extract at 2.5 g/L on GTase activity of S. mutans.Additional file 2: Supplementary Table 1. Synergistic inhibitory activity of extract combination, Glycyrrhizae Radix extract and Rubi Fructus extract, against the biofilm formation of S. mutans with checkerboard assay. This table shows the results for all tested concentration combination of Glycyrrhizae Radix extract and Rubi Fructus extract. After quantitatively analyzing the amount of biofilm with crystal violet, the relative value was calculated by comparing it with the control value. The control value was the amount of biofilm produced without any treatment. The values were calculated from three independent experiments. The shaded cell is a concentration condition that synergistically inhibited biofilm formation. Supplementary Table 2. Synergistic inhibitory activity of compound combination, glycyrrhizin and ellagic acid, against the biofilm formation of S. mutans with checkerboard assay. This table shows the results for all tested concentration combination of glycyrrhizin and ellagic acid. After quantitatively analyzing the amount of biofilm with crystal violet, the relative value was calculated by comparing it with the control value. The control value was the amount of biofilm produced without any treatment. The values were calculated from three independent experiments. Shaded cells are concentration conditions that synergistically inhibited biofilm formation.


Huntington’s disease is a neurodegenerative disorder involving psychiatric, cognitive and motor symptoms. Huntington’s disease is caused by a tandem-repeat expansion in the huntingtin gene, which is widely expressed throughout the brain and body, including the gastrointestinal system. There are currently no effective disease-modifying treatments available for this fatal disorder. Despite recent evidence of gut microbiome disruption in preclinical and clinical Huntington’s disease, its potential as a target for therapeutic interventions has not been explored. The microbiota–gut–brain axis provides a potential pathway through which changes in the gut could modulate brain function, including cognition. We now show that faecal microbiota transplant (FMT) from wild-type into Huntington’s disease mice positively modulates cognitive outcomes, particularly in females. In Huntington’s disease male mice, we revealed an inefficiency of FMT engraftment, which is potentially due to the more pronounced changes in the structure, composition and instability of the gut microbial community, and the imbalance in acetate and gut immune profiles found in these mice. This study demonstrates a role for gut microbiome modulation in ameliorating cognitive deficits modelling dementia in Huntington’s disease. Our findings pave the way for the development of future therapeutic approaches, including FMT and other forms of gut microbiome modulation, as potential clinical interventions for Huntington’s disease. et al. report that faecal microbiota transplant from wild-type into Huntington’s disease mice positively modulates cognitive outcomes, particularly in females. They demonstrate sexually dimorphic and progressive gut dysfunction, and microbial disruption, and provide the first evidence supporting microbiota manipulation as a therapeutic intervention for Huntington’s disease, particularly cognitive symptoms.Gubert   Recent studies have also investigated FMT as an approach for brain disorders including autism spectrum disorder21 and Alzheimer’s disease.22Faecal microbiota transplant (FMT) from healthy donors has been shown to be an effective approach for the treatment of some diseases that affect the GI tract and that have gut microbial disruption as a pathological feature, such as We hypothesize that interventions that ameliorate gut microbiome disruption will, in turn, be therapeutic in Huntington’s disease. In this study, we investigated whether FMT from wild-type (WT) into Huntington’s disease mice will be therapeutic, an approach that has shown promise for some other brain disorders but has never been tested in Huntington’s disease. We assessed FMT impacts on the onset and progression of various aspects of the disease in the R6/1 transgenic mouse model of Huntington’s disease.Male R6/1 hemizygous mice  after back-crossing onto the CBAxC57Bl/6 background for more than 10 generations (generating the CBAxC57Bl/6 strain background) were crossed with female CBAxC57Bl/6 F1 mice to generate male and female WT and R6/1 (Huntington’s disease) littermates. Males and females were bred and housed separately, and therefore, sex was not added as a factor of the study. Genomic DNA from a tail biopsy was used to characterize genotypes. Due to their coprophagic nature, mice were housed according to genotype, sex and treatment to avoid any sharing of microbiota as ingestion of cage-mate faeces can modulate the gut microbiota (2–5 mice per cage). Mice were housed in open-top cages (34 × 16 × 16 cm) with basic sterilized wood shavings and facial tissues for bedding and nesting materials. Cages are sterilized through a washer at 82°C before use.ad libitum access to sterilized food and filtered water (through a 0.5 μm filter) and were housed in a room with a 12:12 h light/dark cycle, controlled for temperature (22°C) and humidity (45%). Cages were changed and body weight assessment was performed weekly. All experiments and procedures were approved by The Florey Institute of Neuroscience and Mental Health Ethics Committee and were performed following the research guidelines and regulations of the National Health and Medical Research Council.All mice had 2, 10% H2, 80% N2), following a previously published protocol with minor modifications.23 Caecal contents were removed from caecal tissue, weighed, resuspended in 4× (w/v) anaerobic PBS, and homogenized by vortexing. The resulting suspension was passed through a Falcon 100 μM nylon cell filter  to obtain the non-fibrous content. The supernatant from all collected caeca was pooled, mixed with an equal volume of 30% anaerobic glycerol–PBS and stored in three Hungate tubes (one tube per day of gavaging to avoid freeze-thaw cycles) at –80°C until required . Prior to oral gavage, pooled caecal supernatant was diluted with 2 × volume of anaerobic PBS (pH: 7.2) resulting in a solution of 5 mg/ml of caecal content.Sex- and age-matched WT littermate mice, naive to behavioural experiments and group housed (with their donor group mates) were used as faecal donors. Donors were culled via cervical dislocation, and caeca were harvested immediately and transferred to 15% glycerol–phosphate-buffered saline (PBS). The subsequent processing was performed under anaerobic conditions (10% CO23 antibiotics (ATBs) were used to induce a disruption of the gut microbiota.24 Mice were randomly assigned into either control (vehicle/vehicle), ATB-only (ATB/vehicle) or ATB/FMT groups  and neomycin sulphate (Sigma-Aldrich) in sterile water at a ratio of 1 mg/ml, for 7 days (at Week 8 of age), following a previously published protocol.24 The vehicle of ATB intervention was drinking water. Non-absorbable ATBs have a relatively small degree of systemic absorption, decreasing possible off-target effects. The FMT intervention consisted of oral administration of 150 µl of the prepared 5 g/ml of donor caeca content solution, for 3 days with 2 days of spacing (at 9 weeks of age). The vehicle control for the FMT intervention was oral administration of 7.5% glycerol–PBS. Plastic needles were used to minimize the effect of the gavage on the mice. The control group received neither ATB nor FMT, only the respective vehicle solution (drinking water and 7.5% glycerol–PBS).To prepare the host for the subsequent FMT,T groups . The ATBFood and water intake were assessed from 6 to 20 weeks of age . To acco25 The Rotarod  consists of a motorized rotating cylinder that is divided into five compartments. One mouse is placed into each compartment, and the rotating cylinder accelerates at a constant speed of 4 rpm before the cylinder gradually accelerates to 40 rpm over 300 s. The time taken for the mice to fall onto the lever below is recorded as an indicator of motor coordination. Mice were tested weekly from 7 to 20 weeks of age after habituation at Week 6 (Rotarod testing was used as an indicator of motor coordination.t Week 6 . Habitua11 from 7 to 20 weeks of age (Mice were assessed weekly for clasping as video a phenotypic characteristic specific to Huntington’s disease transgenic mice (relative to their WT littermate controls) independent of motor coordinations of age . Claspin26  for the assessment of gait and locomotion26 . Mice arAnalysis software was used to determine when individual paws are in contact with the belt of the treadmill to calculate several gait parameters. The gait parameters analysed were propelling time (the duration between maximum paw contact to the start of the swing phase) and braking time (the duration between the beginning of the swing phase and returning to maximum paw contact with the belt). The propel-to-brake ratio was then calculated.27  with stainless steel rod floors. The chambers contained either a patterned background and paper towel bedding or plain background and clean bedding, the same type used in the home cages, to create different environments. For fear conditioning, a conditioned stimulus (CS) of an auditory tone  was paired with an unconditioned stimulus (US) of a foot shock . The mice were habituated for 2 min, while their freezing (locomotor immobility) was measured (this period was considered the conditioning baseline), then the mice received six paired CS–US with the tone lasting 10 s and co-terminating with a 1 s foot shock. The inter-trial interval was 110 s. Freezing was analysed during the 9 s before the onset of the foot shock to ensure the freezing was a conditioned response to the tone. Following the last presentation of the tone, the mice were left within the chamber for 2 min before being returned to their home cages.At 13 weeks of age, the mice underwent fear conditioning to assess their learning capabilities in concurrence with extinction learning, testing longer-term associative cognitive function28 . Fear coThe following day their memory of the CS was tested in a different environmental context within the experimental chamber, to ensure the chamber environment was not paired with the tone in the CS. Mice were allowed to habituate in the chamber for 2 min, while their freezing measurement was analysed (this period was considered the extinction baseline) before being exposed to 45 presentations of the 10 s tone without the shock with an inter-trial interval of 10 s. Percentage freezing during the 10 s tone presentation was analysed and presented as averages of nine blocks of five CSs to track extinction learning. The recorded freezing of the mice during fear conditioning and extinction learning was analysed using VideoFreeze (Med Associates Inc.).17 At 12 weeks of age, mice were single-housed in a sterile cage for 1 h and the number of excreted pellets was recorded as faecal output. These pellets were collected, and the total weight was recorded before being dried at 95°C for >3 h.17 The difference between the initial faeces weight and the dry weight was recorded as a percentage and taken as the faecal water content.We analysed faecal water content as a measure of GI water absorption and faecal output and gut transit time markers of gut function and motility.At 12 weeks of age, non-fasting mice were gavaged with non-absorbable carmine red dye (Sigma-Aldrich), prepared as a 6% (w/v) dilution in 0.5% methylcellulose (Sigma-Aldrich), autoclaved and filtered before administration. Mice were single-housed, and the time taken from gavage to the first appearance of carmine red was recorded as the GI transit time.30 Blood was collected via cardiac puncture 4 h after administration and immediately transferred to an EDTA collection tube and centrifuged at 1000 × g for 10 min. Plasma was then collected, and fluorescence was quantified at an excitation wavelength of 485 nm and an emission wavelength of 528 nm . FITC-Dextran serially diluted in PBS was used to calculate a standard curve.At 14 and 20 weeks of age, mice were fasted for 4 h, then orally gavaged with 150 μl of 4 kDa fluorescein isothiocyanate (FITC) dextran (Sigma-Aldrich) dissolved in PBS to a concentration of 100 mg/ml to measure intestinal epithelial barrier permeability.7 At 14 and 20 weeks of age, mice were euthanized by cervical dislocation; the intestines were removed and placed on a non-absorbent surface, and the length of the caecum and colon was measured using a ruler. The caecum was then weighed and normalized to body weight (g).Gut macroscopy, including caecum and colon measurements, was assessed as an indicator of general gut health.31 Briefly, 20 µl of plasma was suspended in 380 µl of water:acetonitrile  containing 4 µM of 4-methylvaleric acid  as internal standard. Samples were vortexed for 30 s and then mixed at 950 rpm for 10 min at 4°C with a thermomixer . Samples were centrifuged at 16 200 × g (Beckman Coulter Microfuge 22R refrigerated microcentrifuge) for 5 min at 4°C, and the supernatant was transferred to fresh LoBind Eppendorf tubes. Stock solutions of all the SCFAs and BCFAs were freshly and individually prepared in 100% aqueous acetonitrile with 1 mM concentration. This solution was further diluted to have concentrations of 50 to 0.1 µM and the resulting solutions were used as the calibrators. The calibrators (40 µl) and sample supernatant (40 µl) were mixed with 20 µl of 200 mM 3-nitrophenylhydrazine  in 50% aqueous acetonitrile and 20 µl of 120 mM 1-Ethyl-3-(3-dime-thylaminopropyl) carbodiimide  in 50% aqueous acetonitrile with 6% pyridine solution. The mixture was reacted at 40°C for 30 min in a thermomixer at 950 rpm. The reaction was stopped using 20 µl of 200 mM quinic acid  dissolved in acetonitrile:water  by further incubating the mixture at 40°C for 30 min in a thermomixer at 950 rpm. Finally, the samples were then spiked with 20 µl of 10 µM isotope-labelled internal standard mix prepared in accordance with the previously described protocol.31 The samples were further diluted to 2 ml with 15% acetonitrile and 1 µl were then injected for LC-MS/MS analysis. Derivatized SCFAs and BCFAs were processed and detected by Metabolomics Australia  using the conditions previously described2 using Agilent 1290 liquid chromatography (LC) system and Agilent Triple quadrupole 6490 mass spectrometer .Mouse plasma samples were taken at 14 and 20 weeks of age and stored at –80°C prior to analysis. The extraction of short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs) was performed using a modified protocol.Proximal colon samples from 14 and 20 weeks of age were stored at –80°C prior to analysis. Samples were sonicated in 300 µl cell lysis buffer (Invitrogen) and refrozen. The thawed suspension was spun for 5 min at 3000 rpm, and the supernatant was assayed for cytokines as per the manufacturer’s recommendations. The ProcartaPlex multiplex Immunoassay (Invitrogen) was used to determine the Th17 panel of cytokines  using Luminex® 200 System. The DC protein assay kit  was used to determine the protein content. Groups that had less than four samples above the limit of the detection were excluded from the analysis. Results were normalized to supernatant protein concentration and are expressed as pg/mg of protein.23Faecal samples were collected at 8 (pre-interventions), 12  and 20  weeks of age . Mice we32 Paired-end sequencing (2 × 300 bp) of indexed amplicon libraries were performed using a MiSeq Reagent Kit v3 and MiSeq system  at the South Australian Genomics Centre (SAGC), Adelaide, Australia. Microbiota profiling was performed based on a subsampling depth of 9604 or 12 777 sequence reads for the male or female cohort, respectively.Extracted DNA was used for amplicon sequencing of the V4 hypervariable region (515–806 bp) of the 16S rRNA gene on a Miseq Illumina platform as previously described.33 Briefly, the DADA2 workflow was used for denoising, quality filtering, chimaera removal and merging of paired-end sequence reads.34 Taxonomic assignment of amplicon sequence variants (ASVs) was performed against the V4 hypervariable region sequences of the SILVA 132 16S rRNA reference database clustered at 97% similarity.35 Alpha diversity  and weighted UniFrac distances were computed using QIIME2. Genus-level relative abundances were used for downstream analysis.Paired-end 16S rRNA gene sequence reads were analysed using QIIME2 (v2.0).post hoc Dunn’s test for three or more groups. Microbiota composition differences between groups were assessed based on weighted UniFrac distances and analysed using permutational ANOVA (PERMANOVA). Correction of multiple testing was performed using the false discovery rate (FDR) method. Sample ordination and analysis were performed using GraphPad Prism 9 or R (package vegan 2.5-7).Non-parametric analyses were performed using the Mann–Whitney test to compare two groups, or the Kruskal–Wallis test with post hoc pairwise comparisons (emmeans R package) (GraphPad Prism (version 9.2.0) was used to plot the graphs, and the data analysis was performed using R studio (version 1.3.1093). Data are presented as the mean ± standard error of the mean (SEM). Linear mixed models (LMMs) were used to analyse the data separately for males and females. Cumulative LMM with Laplace approximation was used for clasping scores that was measured on an ordinal scale. For covariates declared significant at the 0.05 significance level, we performed a package) .In the LMM cage, effects were specified as random effects in LMM due to the potential effect of coprophagy. For single time point variables (e.g. brain weight), genotype and treatment  were specified as fixed effects. For Y-maze, arm type  was added as fixed effect. For repeated measurement variables (e.g. body weight), time was also added as a fixed effect with the analysis being conducted from Week 8, when interventions started. For contextual fear conditioning (CFC), baseline CFC conditioning and baseline CFC extinction were added as fixed effects, respectively. We considered all two-way interactions between fixed effects when there were at least four samples in each group combination, otherwise only the main effects were considered .https://github.com/SarithaKodikara/Fecal-microbiota-transplant-therapeutic-effect-in-a-Huntington-s-disease-mouse-model.All data, including 16S rRNA amplicon sequencing data, will be made fully available upon publication. Raw sequence data are deposited in the public repository Sequence Read Archive under the BioProject accession number PRJNA795813. The data analysis R code is available at P < 0.0001) (P = 0.01) and Huntington’s disease females (P = 0.003) over time (P < 0.0001). In Huntington’s disease mice, ATB increased body weight (P = 0.0002) and decreased food intake (P < 0.0001). ATB/FMT increased body weight in females over time (P = 0.005), and there was a trend to increase body weight in males over time (P = 0.052), as well as a significant increase in food intake in males (P = 0.0004). Conversely, ATB/FMT induced a decreased food intake (P = 0.0002). Both male and female Huntington’s disease mice showed an increase in water intake over time , while ATB increased the water intake compared with the no treatment (no FMT nor ATB) mice  over time  , despitever time . In malever time .P < 0.0001 for both) (P < 0.0001 for both) (P = 0.0116), ATB and ATB/FMT were not able to modulate either rotarod or clasping performance. DigiGait analysis (assessment of gait and locomotion) in males only revealed an increase in propel:brake ratio in Huntington’s disease mice (P = 0.004) and a decrease in ATB-treated Huntington’s disease mice (P = 0.01) (P = 0.003), with no effect of ATB or ATB/FMT  . For botor both) , as wellor both) . Apart f = 0.01) . Finally ATB/FMT .P = 0.04 and P = 0.004, respectively)  . Interesreatment . On the P < 0.0001) indicating that the CS and US pairing induces conditioning learning (P = 0.0002), as well as an interaction between the baseline and ATB/FMT, overall decreasing the percentage of freezing, suggesting a positive effect of FMT on basal anxiety. At the extinction trial, we saw a decrease in the percentage of freezing in both males and females by time , indicating that in general, the extinction occurred in the test (P < 0.0001) and females (P = 0.0005) over time compared with WT. We also observed an effect at the baseline time point in both males and females (P < 0.0001) with an increased percentage of freezing, while an interaction demonstrated a decrease in freezing over time in both males and females . Also, in female Huntington’s disease mice only, we observed a decrease in freezing behaviour at the baseline time point (P = 0.0005) and an interaction between ATB/FMT and the baseline (P = 0.009). ATB in females increased the percentage of freezing over time (P = 0.01). Post hoc analysis indicated that in males, there was a decrease in extinction in Huntington’s disease mice subjected to ATB/FMT when compared with WT mice subjected to ATB/FMT . In males we saw a trend towards ATB increasing faecal output in Huntington’s disease only (P = 0.056), while ATB increased the faecal output of both WT and Huntington’s disease females (P = 0.01) (P = 0.04). ATB increased the faecal water content in Huntington’s disease males (P < 0.0140) while ATB/FMT over time induced a decrease in faecal water content in both males (P = 0.03) and females (P = 0.01) (P = 0.054), no differences were observed at Week 14 or 20 for either sex  in Huntington’s disease mice were observed, when compared with WT  was detected in both male and female Huntington’s disease mice compared with WT littermate controls   and late (20 weeks of age) stages of the disease, while assessing the effect of ATB and ATB/FMT on these outcomes. Despite observing an increased food and water intake in Huntington’s disease mice compared with WT, both male and female Huntington’s disease mice showed a decreased faecal output, an indirect measurement of constipation as they aged ( = 0.01) . Hunting = 0.01) . While tther sex . Since a with WT , consistctively) . ATB andP = 0.008) (P = 0.03)  . Apart f = 0.03) , we did  = 0.03) . AltogetP = 0.002) (P = 0.007) (P = 0.0002) (P = 0.613) and Faith’s phylogenetic diversity (P = 0.779), as well as the gut microbiota composition (PERMANOVA P = 0.634), were similar between Huntington’s disease and WT females.In determining the potential for early-stage prevention of gut microbiome disruption (via FMT intervention) prior to the onset of gut dysfunction, the gut microbiota of male and female Huntington’s disease mice were characterized at 8 weeks of age, using faecal 16S rRNA sequencing and bioinformatics. Reductions in faecal total bacterial load , and cha= 0.002)  and dive= 0.007) , while t 0.0002) . In contP = 0.042 and P = 0.034, respectively). These results were consistent with significantly larger microbiota variation within Huntington’s disease mice compared with the WT mice at Week 8   was significantly lower compared with those of WT littermate controls (PERMDISP ctively) , with a P > 0.05)  when compared with the levels in the untreated WT group  . In femaWT group . SimilarP = 0.005 and P = 0.007, respectively) (P = 0.004 for both WT and Huntington’s disease genotypes)  but not in Huntington’s disease males   (P > 0.05). Together, these results suggest that WT gut microbiota engraftment in Huntington’s disease mice was more effectively established in females compared with males.For both WT and Huntington’s disease males that received ATBs, reductions in the number of species observed (ctively)  and compnotypes)  were obs= 0.004) . At 20 w= 0.004) , and com= 0.040) . In contP = 0.04) (P = 0.04) (P = 0.04)  . At 20 w = 0.04)  and an e = 0.04) . We did  = 0.04) .P = 0.02) (P = 0.059) (P = 0.059) (P = 0.04) (P = 0.01)   and, at = 0.059) . In fema= 0.059) . However = 0.04) , and IL- = 0.01) . No sign = 0.01) .This is the first study to investigate the therapeutic potential of FMT as a means to shift the recently discovered pathological Huntington’s disease gut microbiome characteristics (dysbiosis) towards a healthy profile. FMT modulated cognitive outcomes in Huntington’s disease, an effect that was more pronounced in females. This study reveals an inefficiency of engraftment of the WT microbiota in Huntington’s disease male mice and an associated sexually dimorphic disruption and instability of the gut microbial community, circulating SCFA levels and gut immune profiles in Huntington’s disease. We report, for the first time, significant gut dysfunction in the late stage of the Huntington’s disease phenotype. Sexual dimorphism was also observed for this trait, with Huntington’s disease males being more affected. Our findings, together with those of others, suggest that Huntington’s disease is not just a brain disorder but rather is a whole-body disease with pathological features including sexually dimorphic and progressive gut dysfunction and microbial disruption. These findings will inform future microbiota-targeted therapies for Huntington’s disease, to address both central and peripheral symptoms of this fatal disease.36 Improvement in learning and memory has also been one of the key targets for the gut microbial interventions for brain disorders.38 Corroborating previous findings,9 we have found reduced novel arm preference in Huntington’s disease mice in the Y-maze cognitive test. GI inflammation and gut microbiome disruption have been shown to impair cognitive performance in the Y-maze,39 and in our study, ATB in female WT mice inhibited cognitive performance in the Y-maze, which was resolved after the FMT intervention. We have also replicated previous findings showing that Huntington’s disease mice exhibit decreased freezing in the extinction trial of the fear conditioning test, indicating impaired fear learning and memory.40 Recent findings have implicated the gut microbiota in fear memory and impaired extinction learning with ATB-treated and germ-free mice showing impaired extinction learning.41 We observed similar results in females with the ATB treatment. In males, we found a decrease in extinction induced by ATB/FMT in Huntington’s disease mice when compared with WT mice. However, in females, the FMT intervention was able to fully rescue the cognitive deficits observed in Huntington’s disease mice. Altogether, these results reflect the sexually dimorphic FMT engraftment observed in Huntington’s disease mice and support the hypothesis that interventions that ameliorate gut microbiome disruption (dysbiosis) could in turn be therapeutic for this disease.The onset of cognitive symptoms in Huntington’s disease is a driving factor in the progressive decline in quality of life.42 All variables besides the genetic and microbiota differences between our donor WT and recipient Huntington’s disease mice, were carefully controlled and kept consistent in the present study. We have also followed the most recent guidelines for reporting and performing animal FMT43 and have used established protocols for both the ATB gut preparation24 and intervention.23 FMT engraftment was successful in both WT males and females, whereas a lower fidelity of engraftment and subsequently, lack of phenotype modulation was observed in Huntington’s disease mice, particularly in Huntington’s disease males. These results suggest the importance of further understanding complex donor-recipient dynamics in Huntington’s disease for the optimization of future clinical intervention strategies associated with modulating microbial–host relationships.Successful engraftment of the gut microbiota is dependent on many factors, including genetic background, composition of the gut microbial community, inflammation status, housing environment, as well as diet of the donor and recipient individuals.45 An unstable state involving increased heterogeneity in microbiome composition over time, which has been associated with numerous negative health outcomes,46 was observed in the present study, particularly in Huntington’s disease males, extending our previous study in unmanipulated R6/1 Huntington’s disease mice.16 Notably, in the present study, we observed that the instability of the Huntington’s disease gut microbiome was concurrent with its resistance to FMT engraftment. Given that the host genetics greatly influences the physical structure of the gut, which then determines the chemical and physical environment inhabited by the gut microbiome,47 one possible explanation is that the expression of mutant HTT (via the human Huntington’s disease transgene) by the Huntington’s disease mice altered the gut mucosal lining and overall architecture, thus exerting stochastic effects on microbiome composition while impeding the colonization of microbes adapted to a healthy gut. This hypothesis should be further investigated.The stability of the gut microbiome is critical to host health, in which complex interactions between microbial species increases resilience of the microbial community to perturbation.48 and SCFAs are considered key regulators of the microbiota–gut–brain crosstalk.49 While increases in SCFA levels are usually associated with beneficial outcomes, increased plasma acetate levels have been shown in multiple sclerosis patients compared with controls, with levels correlating with greater disability and increased T helper 17 (TH17) + cells.50 Interestingly, acetate is considered to be an important metabolite for host resistance to bacterial infection, having a pivotal role in the pro- and anti-inflammatory balance, with high acetate promoting superior immune control.52 The gut immune profile is crucial for the host gut microbiota composition,53 and it has been recently shown to be a key modulatory factor for the efficacy of FMT in C. difficile infection.54 We have found increased IL-7R colon levels in Huntington’s disease males, while Huntington’s disease females showed a decrease in IFNγ colon levels. Intestinal IL-7R signalling from the TH17 cell subset is present in inflamed colon tissue, closely related to inflammatory bowel disease.55 IFNγ mediates the recognition and response to bacteria,56 both key players in the host bacterial defence and in shaping the gut microbial community. The imbalance of the acetate levels and intestinal immunity observed in male and female Huntington’s disease mice  could also be related to the sexual dimorphism observed in the FMT engraftment in Huntington’s disease. Further studies are needed to test this hypothesis. Nevertheless, our results raise opportunities to improve FMT efficacy for therapeutic intervention for both sexes in Huntington’s disease by focusing on modulating gut microbial stability, acetate levels and immune profiles.Male Huntington’s disease mice showed increased levels of acetate (a key SCFA) in plasma at early and late stages of the disease. The major source of circulating SCFAs is the fermentation of indigestible carbohydrates by intestinal bacteria,In conclusion, our results demonstrate a potential role for gut microbiota in modulating cognitive outcomes in Huntington’s disease, presenting opportunities to pursue novel microbiota-targeted therapies for this devastating disease in future preclinical studies and clinical trials.fcac205_Supplementary_DataClick here for additional data file.


It is important for patient safety to assess if international changes in perioperative care, such as the focus on venous thromboembolism (VTE) prevention and minimally invasive surgery, have reduced the high post colectomy VTE risks previously reported. This study assesses the impact of changes in perioperative care on VTE risk following colorectal resection.This was a population‐based cohort study of colectomy patients in England between 2000 and 2019 using a national database of linked primary  and secondary  care data. Within 30 days following colectomy, absolute VTE rates per 1000 person‐years and adjusted incidence rate ratios (aIRRs) using Poisson regression for the per year change in VTE risk were calculated.Of 183 791 patients, 1337 (0.73%) developed 30‐day postoperative VTE. Overall, VTE rates reduced over the 20‐year study period following elective  but not emergency surgery. Similarly, yearly changes in VTE risk reduced following minimally invasive resections  but not following open resections. There was a per year VTE risk increase following open emergency malignant resections .Yearly VTE risks reduced following minimally invasive surgeries in the elective setting yet in contrast were static following open elective colectomies, and following emergency malignant resections increased by almost 2% per year. To reduce VTE risk, further efforts are required to implement advances in surgical care for those having emergency and/or open surgery. Perioperative care has changed with an increase in venous thromboembolism (VTE) prevention. VTE rates have reduced following minimally invasive but not open colectomies by 4%–7% annually. However, rates post emergency malignant resections have increased by almost 2% annually, suggesting future focus on improving postoperative emergency colectomy VTE rates is needed.Colorectal resection, the second most common major abdominal operation in the developed world, forms a substantial proportion of resectional abdominal surgery . In the Within the last two decades colorectal surgical care has changed. Globally VTE became a significant postoperative complication with multiple high‐quality studies devoted to investigating methods of reducing its risk following colorectal resection , 5, 6, 7In addition to VTE guidance, minimally invasive resection has been increasingly adopted for benign and malignant colorectal disease in developed and developing nations globally , 23, 24.The focus on VTE prevention, changes in operative techniques and ERAS will probably have altered VTE risk following colectomy, yet this has not been established. This study aims to quantify variations in VTE rates over the last 20 years in relation to international changes in perioperative care following colorectal resection.The study was approved by the Independent Scientific Advisory Committee approval board (Protocol 19_180RA3).This study utilized three validated and linked healthcare databases that have been described previously , 35, 36.The cohort of patients undergoing colorectal resections between the years 2000 and 2019 was identified using OPCS‐4 codes Appendix  from HESCompletely endoscopic operations and those confined to the anal canal were excluded, including patients aged <18 years old at the date of operation. Patients identified as having a VTE event prior to resection were also excluded due to their inherently increased risk of VTE . PreviouCalendar year was the main exposure of interest, defined as a continuous variable and as a binary variable pre‐ and post‐2010 (the year NICE guidance [CG92] from England advocating extended VTE prophylaxis in CRC resections was published). Other exposures were selected from previous literature as follows , 45. SurNational NICE guidance from England on postoperative VTE prevention, published in 2010, stated that those undergoing abdominal surgery at risk of VTE should receive combined mechanical and chemical VTE prophylaxis during admission and those undergoing cancer surgery should receive an extended 28 days of postoperative VTE chemoprophylaxis . TherefoThe primary outcome was a VTE event diagnosis, either a pulmonary embolism or a peripheral deep vein thrombosis, defined from medical and ICD‐10 codes in the linked CPRD and HES datasets 30 days post resection. VTE events were only considered valid if supported by either a prescription for any anticoagulant medication from the British National Formula licensed to treat a VTE in the UK or other evidence of treatment within an anticoagulation clinic  between 15 days before and 90 days after VTE diagnosis, or a date of death within 30 days of the event. This time frame was used to allow for anticoagulant prescribing in the community to follow on from anticoagulation clinic or inpatient prescribing for a VTE event. Furthermore, VTE as an underlying cause of death was included as evidence of VTE diagnosis. A 30‐day postoperative follow‐up was selected because this is the period of time required for the intervention of extended VTE prophylaxis and is a standard follow‐up end‐point in other national population‐based cohort studies, allowing ease of comparison  and usedCohort demographics were presented as proportions and stratified by admission type and surgical indication. 30‐day post colectomy incidence risks, presented as percentages, and absolute VTE incidence rates (IR) per 1000 person‐years were calculated for each year of the study period and stratified a priori by admission type and surgical indication , 16, 44.P < 0.1 using a generalized likelihood ratio test, was assessed within the multivariable model to test for any interactions between linear trend by year of colectomy and admission type, surgical indication and operative technique.Poisson regression was used to calculate adjusted incidence rate ratios (aIRRs) for linear trend in year‐on‐year VTE risk, adjusted for all other exposures within the analyses and where relevant the relative change in VTE risk as a percentage over the 20‐year study period. Effect modification, significance set at In addition, a sensitivity analysis examining whether there was an interaction between the linear trend in VTE rates by year and pre‐ and post‐2010, the year NICE guidance on extended VTE prophylaxis use post malignant colectomy was published, was conducted.® version 16.1 (StataCorp LLC).All data management and analyses were performed using Stata SEOverall, 183 791 patients in the validated cohort underwent colorectal resection and 1337 (0.73%) developed VTE within 30 days after surgery with an absolute IR of 90.67 (95% CI 85.94–95.66) per 1000 person‐years. Most patients were of white ethnicity (91.19%) and 93 598 (50.93%) were male.P < 0.0001, χ2 test for trend; see Figure P < 0.0001, Kruskal–Wallis test; see Table There was an increase in minimally invasive surgery over time. Between 2000 and 2019 the yearly proportion of colectomies performed minimally invasively increased from 0.59% to 73.31% in the elective setting and from 0.36% to 26.17% in the emergency setting  per 1000 person‐years (0.54%) and overall significant relative VTE risk reduction of 31.25% (95% CI 5.69%–49.88%) over the 2000–2019 study period . There were also significant interactions between the linear trend in VTE risk and both admission type, Following elective benign and malignant minimally invasive colorectal resections a 6.78% and 5.62% year‐on‐year reduction in VTE risk was observed  with 30‐day VTE IR per 1000 person‐years of 32.57  and IR 44.51  respectively. Following elective benign or malignant open resections no significant year‐on‐year VTE risk reduction was observed  with 30‐day VTE IR per 1000 person‐years of 69.92  and IR 88.88  respectively.In the emergency setting, patients undergoing minimally invasive benign colorectal resections had a significant year‐on‐year reduction in VTE risk of 4.24%  with a 30‐day VTE IR of 58.89 (95% CI 39.47–87.86) per 1000 person‐years (0.48%). No significant year‐on‐year reduction in VTE risk was observed in emergency malignant minimally invasive resections  with a 30‐day VTE IR of 133.98 (95% CI 86.44–207.66) per 1000 person‐years (1.08%). In patients undergoing emergency open colorectal surgery, those undergoing cancer resection had a significant 1.89% year‐on‐year increase in VTE risk  with a 30‐day VTE IR of 185.52 (95% CI 164.84–208.79) per 1000 person‐years (1.43%). In benign emergency open resections, no significant year‐on‐year increase in VTE risk was seen  with a 30‐day VTE IR of 130.51 (95% CI 117.63–144.80) per 1000 person‐years (0.99%).P = 0.9305, was found between year‐on‐year VTE trend post colorectal resection and pre/post the year 2010, the year NICE guidance on extended VTE prophylaxis use was published, with no overall significant step change in VTE risk between pre‐ and post‐2010 observed . No change in effect modification or step change in VTE risk was observed using 1‐ or 2‐year washout periods post the introduction of NICE guidelines. This was despite the overall reduction in VTE rates seen in elective colorectal resections that began prior to the year 2010  to 6 (IQR 4–11) days over 2000–2019. Therefore, our study's finding of a year‐on‐year reduction in VTE risk following elective colorectal resection may be associated with the improved perioperative care ERAS patients receive, in addition to more minimally invasive surgery and extended VTE prophylaxis guidelines . FurtherFollowing emergency surgery, we found an annual reduction in VTE risk following minimally invasive benign resections and a year‐on‐year increase in VTE risk post open malignant colorectal resections. This contrasts with contemporary data reporting no differences in annual VTE risk following emergency colectomy . AlthougThere was an increase in year‐on‐year VTE risk following open emergency malignant colorectal resections despite guidance supporting extended VTE prophylaxis in this subgroup. However, the ENDORSE study highlighted that emergency compared to elective surgical patients were less likely to receive appropriate VTE prophylaxis . Other pOver the last 20 years VTE risk following elective colorectal resections has reduced in absolute terms, specifically when compared to the VTE risk following emergency resections. This risk reduction is most probably due to changes in perioperative surgical care. This suggests future focus on improving postoperative emergency colectomy VTE rates is needed with the application of specific advances observed in the elective setting, such as emergency ERAS pathways utilizing minimally invasive techniques where possible.All authors meet the conditions for authorship, CRediT. CLL: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Writing—Original draft, Writing—Review and editing, Visualization, Project administration. CC: Conceptualization, Methodology, Software, Validation, Resources, Writing—Review and editing, Visualization, Supervision, Project administration. JW: Software, Validation, Resources, Writing—Review and editing, Visualization, Supervision. OP: Validation, Writing—Review and editing, Visualization, Supervision. DH: Conceptualization, Methodology, Software, Validation, Investigation, Resources, Data curation, Writing—Review and editing, Visualization, Supervision, Project administration.There are no conflicts of interest, use of off‐label or unapproved drugs or products and use of previously copyrighted material to declare.The study was approved by the Independent Scientific Advisory Committee approval board (Protocol 19_180RA3). Data from Hospital Episode Statistics and Clinical Practice Research Datalink if used as instructed, as the authors have, do not require individual specific patient consent and this is implied under the study approval. All material is of the authors’ original creation and there is no clinical trial registration required.Appendix S1Click here for additional data file.


The impacts of the current COVID‐19 pandemic on maternal and foetal health are enormous and of serious concern. However, the influence of SARS‐CoV‐2 infection at early‐to‐mid gestation on maternal and foetal health remains unclear.Here, we report the follow‐up study of a pregnant woman of her whole infective course of SARS‐CoV‐2, from asymptomatic infection at gestational week 20 to mild and then severe illness state, and finally cured at Week 24. Following caesarean section due to incomplete uterine rupture at Week 28, histological examinations on the placenta and foetal tissues as well as single‐cell RNA sequencing (scRNA‐seq) for the placenta were performed.Compared with the gestational age‐matched control placentas, the placenta from this COVID‐19 case exhibited more syncytial knots and lowered expression of syncytiotrophoblast‐related genes. The scRNA‐seq analysis demonstrated impaired trophoblast differentiation, activation of antiviral and inflammatory CD8 T cells, as well as the tight association of increased inflammatory responses in the placenta with complement over‐activation in macrophages. In addition, levels of several inflammatory factors increased in the placenta and foetal blood.These findings illustrate a systematic cellular and molecular signature of placental insufficiency and immune activation at the maternal–foetal interface that may be attributed to SARS‐CoV‐2 infection at the midgestation stage, which highly suggests the extensive care for maternal and foetal outcomes in pregnant women suffering from COVID‐19. Here, we report the follow‐up study of a pregnant woman of her whole infective course of SARS‐CoV‐2, from asymptomatic infection at gestational week 20 to mild and then severe illness state, finally cured at Week 24 and delivering at Week 28 due to incomplete uterine rupture. The placenta from this case exhibited more syncytial knots and lowered expressions of syncytiotrophoblast‐related genes. The single‐cell RNA sequencing analysis demonstrated impaired trophoblast differentiation, activation of antiviral and inflammatory CD8 T cells, as well as the tight association of increased inflammatory responses with complement over‐activation in macrophages. These findings illustrate a systematic cellular and molecular signature of placenta insufficiency and immune activation that may be attributed to SARS‐CoV‐2 infection at midgestation stage, which highly suggests the extensive care for maternal and fetal outcomes in COVID‐19 pregnancy. Although pregnant women were more susceptible to the coronaviruses and several pregnant women infected with SARS‐CoV‐2 are reported to have severe adverse outcomes,The placenta expresses the receptors for SARS‐CoV‐2, including ACE2 and TMPRSS2, throughout pregnancy,Here, we reported a case of a COVID‐19 pregnant woman (marked as COVID‐RS in the following) infected with SARS‐CoV‐2 at the 20th week of gestation. We followed up the woman from asymptomatic, mild illness and severe illness to recovery stages until pregnancy termination due to an incomplete uterine rupture at gestational week 28. Histological analysis of the foetal and placental tissues as well as single‐cell transcriptome for the placenta was performed. No intrauterine transmission of SARS‐CoV‐2 was found in this patient, whereas the cellular and molecular signatures indicate placenta insufficiency and significant antiviral and pro‐inflammatory response at the maternal–foetal interface. The findings thus highlight the extensive care for maternal and foetal health in pregnant women suffering from COVID‐19, even after timely clinical treatment for the virus infection.22.1The study was approved by the Ethics Committee Medical Ethics Committee at The Third Affiliated Hospital of Guangzhou Medical University . Written informed consent for this study was obtained from the enrolled women.2.2Nasopharyngeal and vaginal swabs were collected at admission and during hospitalization to test for SARS‐CoV‐2. At delivery, maternal blood, umbilical cord blood and foetal blood were collected in EDTA tubes. Full‐thickness placental biopsies were obtained. In the case of caesarean delivery, amniotic fluid was collected without blood or meconium contamination. Tissue samples from the foetus were collected after death with written informed consent from the pregnant woman. Both biopsies and blood samples were obtained sterilely by a dedicated operator. The demographic characteristics of this COVID‐RS case and the gestational age‐matched noninfection preterm patients  enrolled in this study are displayed in Table 2.3For single‐cell RNA‐seq, cells from the placenta were isolated by using the previously described protocol.2.4https://github.com/plger/scDblFinder) to eliminate doublets/multiplets in single‐cell sequencing data according to the previous reports.For placenta samples, 5′ and 3′ transcriptome sequencing data from the COVID‐RS patient and 3’ transcriptome sequencing data from 2 CTRL women with the comparable duration of gestational age were analysed. Seurat v.3 was used for downstream analysis. The following criteria were then applied to each cell of all samples: gene number between 200 and 6000 and mitochondrial gene percentage proportion < 0.05. We utilized the R package scDblFinder2.5p < 0.05. Gene Ontology (GO) enrichment analyses of differentially expressed genes were implemented by the ClusterProfiler R package. GO terms with corrected p‐value < 0.05 were considered significantly enriched by differentially expressed genes. Dot plots were used to visualize enriched terms by the enrichplot R package.Wilcox in Seurat v.3 (FindAllMarkers function) was used to perform differential gene expression analysis. For each cluster of placenta cells, differentially expressed genes (DEGs) of COVID‐RS placenta were generated to compare with the same cell type of the CTRL group. A gene was considered significant with adjusted Bonferroni‐adjusted 2.6https://github.com/Teichlab/cellphonedb) used the cell type annotation and counts from our single‐cell transcriptomics data to compute cell–cell communication within the identified cell subtypes between COVID‐RS case and CTRL group. The default ligand–receptor pair information was used in this process. The p value < 0.05 indicated significant enrichment of the interacting ligand–receptor pair in each of the interacting pairs of cell subpopulations and Cellchat was used as a systematic analysis of cell communication based on the network analysis and pattern recognition approaches.CellPhoneDB2.7https://github.com/PierreBSC/Viral‐Track).To systematically scan for viral RNA in scRNA‐seq data, an Unsupervised Viral‐Track Pipeline was used2.82O2. The sections were counterstained with haematoxylin and mounted. CD68 were co‐staining with Anti‐C5aR. For frozen tissue, 10‐μm transverse sections were used for immunofluorescence (IF) using Rabbit anti‐Human C3c Complement/FITC  and Anti‐Cytokeratin 7 antibody (Table t test.For haematoxylin and eosin (HE) staining and immunohistochemistry (IHC), tissue samples were fixed with 10% buffered formalin at room temperature for 6 h. Samples were embedded in paraffin. After routine rehydration and antigen retrieval, 5‐μm paraffin sections were stained for HE or with the primary antibodies Table . The secdy Table . Negativ2.92O2 to quench endogenous peroxidases for 10 min at room temperature. Washed slides were boiled in RNAscope Target Retrieval Reagents for 15 min. The barriered slices were then incubated for 30 min in RNAscope Protease Plus at 40℃. The v‐nCov2019‐S probe , positive probe  and negative probe  were designed and synthesized by ACD. Tissues were counterstained with haematoxylin. After tapping off the excess counterstain, the slides were mounted and viewed under a microscope.The RNAscope 2.5 Assay for in situ hybridization (ISH) from Advanced Cell Diagnostics (ACD) was performed according to the manufacturer's instructions. In brief, FFPE‐embedded tissue sections were baked on the slides at 60°C for 2 h. Each section was treated with H2.10Whole lysates from tissues were extracted with RIPA buffer containing protease inhibitor cocktail (Sigma‐Aldrich). Protein concentrations were determined using the BCA™ Protein Assay Kit (Pierce). Western blotting was performed as described previously. Antibodies used for Western blotting include ACE2, hCG beta and β‐actin Table .2.11Inflammation 20‐Plex Human ProcartaPlex™ Panel  was used to assess the concentration of cytokines in maternal and foetal plasma and placenta samples from the COVID‐RS case and CTRL group according to the manufacturer's protocol via Luminex MAGPIX system (Luminex).33.1+4 weeks by ultrasound evaluation. On May 29 (27+6 weeks of gestation), a routine throat swab test for SARS‐CoV‐2 on this woman showed re‐positive while without any symptoms of COVID‐19. However, her throat swab test for SARS‐CoV‐2 was negative on June 2.The whole process of the infection and treatment of a pregnant COVID‐19 case was illustrated in Figure +4 weeks of gestation), she was admitted to the Emergency Department of the Third Hospital of Guangzhou Medical University due to persistent lower abdominal pain. Considering the short interpregnancy interval (IPI) of this pregnancy from her previous caesarean section (seven months) and the emergent clinical presentations, the uterine rupture was highly suspected. Termination of pregnancy was conducted following an examination and the patient's consent. Ultrasound graph showed severe foetal distress, normal placenta location and amniotic fluid volume before the caesarean section. Surgical observation showed an incomplete uterine rupture in the uterus wall around the previous caesarean incision, where muscle layer was lacking, and only uterine serosa remained and bulged outward. The foetus weighed 920 g, while died during caesarean delivery due to severe asphyxia. None apparent morphologic abnormality was observed in the foetus, placenta and umbilical cord at delivery.On June 4 (283.2http://virusite.org/) by using Viral‐Track revealed no SARS‐CoV‐2‐related reads in the placenta cells of this case.To assay SARS‐CoV‐2 infection, whole maternal blood, cord blood and amniotic fluid collected during caesarean delivery were sent for serological and virological tests. IgM to SARS‐CoV‐2 in the maternal and cord blood was negative. IgG to SARS‐CoV‐2 in maternal and cord blood tested positive, with the cut‐off index (COI) 8.04 and 4.4 respectively. The virological tests of SARS‐CoV‐2 by qRT‐PCR showed negative in all samples, including the maternal blood, cord blood, amniotic fluid, placenta and foetal tissues . RNAscope in situ hybridization (ISH) for SARS‐CoV‐2 RNA was carried out in the placenta and foetal tissues, and no viral RNA puncta signal was detected in these tissues Figure , which wn = 3, details described in Table Histological examinations on the placenta and foetal tissues revealed relatively sparse villi and normal morphology of foetal organs, including the brain, lung, colon, liver and fallopian tube Figure . Using p3.3To illustrate the molecular alterations in the maternal–foetal interface with SARS‐CoV‐2 infection at the midgestation stage, scRNA‐seq analysis was performed on the COVID‐RS placenta  and two CTRL placentas using droplet‐based 10X Genomics platform Figure . Followi3.4KRT7 and PERP , and the hormone biosynthesis genes, such as CGA and CSH2, were decreased in the COVID‐RS placenta , known to significantly potentiate SARS‐CoV‐2 infectivity, displayed higher expression in dMP, dM2, HB, Endo and VCT1 subsets from COVID‐RS placenta than that from CTRL placenta  analysis showed multiple molecular changes of trophoblastic features upon SARS‐CoV‐2 infection. Compared with the CTRL group, immune tolerance–associated genes, including a Figure . Interesn Figure . Howevera Figure . In addig Figure . Althougg Figure . Neuropia Figure , which s3.5IFITM1, IRF1, IRF2, JAK1, OASL, SAMHD1 and GBP2 were upregulated in CD4T, CD8T, NK1, NK2 and monocyte subsets from COVID‐RS, and STAT1, IRF2, JAK1, SAMHD1 and GBP2 were upregulated in macrophage subsets including foetal‐derived HB cells from COVID‐RS , IL‐8 and MCP‐1 (encoded by CCL2) in the COVID‐RS placenta , MIP‐1 beta (encoded by CCL4), CD62E, CD62P and ICAM‐1 in cord blood from the COVID‐RS case compared to the corresponding CTRL .4Here, we reported a whole COVID‐19 disease process in a pregnant woman infected with SARS‐CoV‐2 at the second trimester, covering stages from asymptomatic, mild illness, severe illness, recovery, to pregnant termination due to an incomplete uterine rupture. Combined with the scRNA‐seq of the placenta, we provided for the first‐time systematic cellular and molecular evidence indicating placental insufficiency and potential impacts on the maternal and foetal health that may be attributed to antiviral and pro‐inflammatory responses induced by SARS‐CoV‐2 infection at the midgestation stage in a recovered COVID‐19 pregnant woman.Recent studies reported that around 13.55%–88% of pregnant women exhibit asymptomatic infection for SARS‐CoV‐2,Although our study and others’ reports demonstrated the expressions of SARS‐CoV‐2 entry receptors,+ T cells, which have a mixed profile of T cells and retain the capacity to respond to pro‐inflammatory events,Meanwhile, we also found decidua CD8HLA‐C and HLA‐G in the EVT subset, which is critical to establish protective immunity at the maternal–foetal interface, are downregulated in COVID‐RS placenta, indicating the impairment in immune tolerance to the foetus. Several key pathways that regulate EVT invasion, such as WNT, PRL and kisspeptin pathways,In this case, we were unable to follow through with the maternal and foetal outcomes until late gestation because the pregnancy was terminated due to uterine rupture caused by a short IPI from the last caesarean section. We also did not find apparent histopathological abnormalities in the foetus from this COVID‐RS case, except for a relatively low foetal weightCXCL8 and IL‐1B, indicating that an inflammatory status was still present in the placenta although SARS‐CoV‐2 was absent. The placental inflammatory response, specifically IL‐1β, induced by viral infections, is known to increase the risk of perinatal developmental abnormalities.Second, the notable pro‐inflammatory state in the COVID‐RS placenta indicates potential harm to foetal outcomes. Macrophages are one of the main leukocyte groups present throughout pregnancy and are a source of homeostasis as well as inflammatory responses to infection.Third, the features of the activated complement system may contribute to triggering the inflammatory response in the COVID‐RS placenta. It has been found that most COVID‐19 patients with respiratory failure present consistent over‐activation of complement systemBy far the evidence on the long‐term outcomes of pregnant women who had suffered from COVID‐19 is relatively scarce. In a study of 675 pregnant women admitted for delivery, postpartum readmission differed among women with symptomatic COVID‐19 (6.7%), asymptomatic infection of COVID‐19 (3.6%) and without COVID‐19 infection (1.5%).Additionally, we recognize that the main limitation of our study is the lack of an appropriate control group. In this study, gestationally age‐matched nonexposed placentas delivered due to cervical incompetence were chosen as the control group. Although the ideal control is the gestationally age‐matched, nonexposed, and with uterine rupture placenta, clinically it is extremely difficult to obtain such controls. Our present findings are consistent with the recent report from Lu‐Culligan's study of the placental transcriptome in COVID‐19 cases, showing significant activation of SARS‐CoV‐2 infection–related immune genes, including complement factors and interferon‐stimulated genes. These studies together indicate local inflammatory response to systemic SARS‐CoV‐2 infection in the placenta of SARS‐CoV‐2‐infected pregnant women.So far, there has been limited evidence regarding the follow‐up of maternal and foetal health in women suffering from COVID‐19, especially being infected at early‐to‐mid gestation. Nonetheless, our findings in the placenta of this case demonstrate defects in trophoblasts and their interactions with other uterine cells, as well as antiviral response, pro‐inflammation response and complement over‐activation at the fetomaternal interface from the recovered COVID‐19 pregnancy. These cellular and molecular changes have been highlighted to affect foetal development and the short‐term or long‐term health of the offspring. Early screening of asymptomatic infection, timely clinical treatment and extensive monitoring for pregnant women should be essential strategies to reduce the harm to the infected mothers and newborns. In addition, a long‐term follow‐up study in a larger population will warrant an in‐depth understanding of the long‐term consequences of COVID‐19 pregnancy and their offspring.There are no competing financial or nonfinancial interests regarding this work.D.J.C, Y.L.W. and B.C. jointly conceived and supervised the project, interpreted the data and revised the manuscript. J.S.C followed up the patient, interpreted the clinical data and participated in draft preparation. L.L.D. and F.Y.W performed scRNA‐seq and data analysis and drafted the manuscript. X.S. and W.Z.Y performed bench analysis of the placenta and participated in data analysis. X.Y.W, D.X.C, X.F. P, R.F.C, F.W.X, X.Y. L, S.P.W, M.L.Z, M.X.L., L.L., H.Y., F.H. and L.Y. managed the patient and collected clinical information. S.S.Z, L.J.H. and Y.L.L helped in sample collection and processing. S.L.B, L.Z.Z and Y.Y.L participated in data analysis. Q.P.J and Z.T.X. performed the pathological examination and clinical data interpretation. All authors have read and approved the final manuscript and declared no conflict interests.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Figure S5Click here for additional data file.Figure S6Click here for additional data file.Figure S7Click here for additional data file.Table S1–S3Click here for additional data file.


Eosinophilic airway inflammation is a recognized inflammatory pattern in subgroups of patients with chronic obstructive pulmonary disease (COPD). However, there are still conflicting results between various studies concerning the effect of eosinophils in COPD patients. Our aim with this study was to evaluate eosinophilic inflammation and its relation to the clinical characteristics in a group of COPD patients.1% predicted < 50 or with ≥ 1 exacerbation leading to hospital admission or ≥2 moderate or severe exacerbation history were consecutively enrolled from outpatient clinics.Stable COPD patients with FEV1% predicted was 35.9 ± 11.3. Eosinophilic inflammation (eosinophil percentage ≥2%) was evident in 54 (60%) of the patients. Participants with eosinophilic inflammation were significantly older and had better FEV1 predicted % values. Eosinophilic COPD patients were characterized with better quality of life and fewer symptoms. COPD patients with noneosinophilic inflammation used supplemental long‐term oxygen therapy (LTOT) more frequently compared to patients with eosinophilic inflammation . Eosinophilic inflammation is associated with less dyspnea severity measured by mMRC  and less LTOT use  regardless of age, severity of airflow limitation, and having frequent exacerbation phenotype.We included 90 male COPD patients, with a mean age of 63.3 ± 9.2. Mean FEV1% predicted.Our study supports the growing evidence for a potential role of eosinophilic inflammation phenotype in COPD with distinctive clinical characteristics. Eosinophilic inflammation is inversely associated with dyspnea severity measured by mMRC and LTOT use independently from age, total number of exacerbations, St. George Respiratory Questionnaire (SGRQ) total score and FEV Most frequently used methodology to identify eosinophilic inflammation in airway diseases is sputum eosinophil percentage.Definition of eosinophilic inflammation in stable COPD patients varies between studies, and there is no consensus on the definition of blood eosinophilic inflammation in the literature yet. Sputum eosinophilia has been observed in approximately 1/3 of stable COPD patients,During acute exacerbation of COPD (AECOPD), reports show that eosinophilic airway inflammation is identified by blood eosinophils in 9.6%–45% of casesEmphysema % shown in computed tomography (CT) at baselineEmerging evidence shows a potential role for eosinophilic inflammation indicating distinctive clinical features in COPD and a potential marker for tailored treatment approach.22.11% pred <50 or (2) ≥ 1 exacerbation leading to hospital admission, or (3) ≥2 moderate or severe exacerbation history. The institutional ethical committee approved the study protocol, and written informed consent was obtained from all participants. Exclusion criteria comprised concomitant asthma, lung malignancy, or exacerbation within 1 month.Designed as a cross‐sectional study based on two pulmonary medicine departments,  outpatient clinic in between February 2016 to February 2017. We consecutively enrolled stable COPD patients with (1) FEV2.21/FVC < 0.70. Airflow limitation was classified according to FEV1% predicted: >80% were defined as GOLD 1, while FEV1% predicted between 80 and 50, between 50 and 30, and below 30 were defined as GOLD 2, GOLD 3, and GOLD 4, respectively. Stable COPD was characterized as requiring no increase in bronchodilator use, no use of oral corticosteroids or antibiotics, no unscheduled doctor visits, or hospitalization over the previous 4 weeks.COPD patients were defined as those with chronic airway inflammation symptoms, history of exposure to risk factors, and fixed airflow limitation in spirometry.2.3AECOPD was defined as an acute event characterized by a worsening of the patient's respiratory symptoms beyond normal variation and leading to a change in medication, while frequent exacerbators were defined as patients experiencing two or more exacerbations per year.2.4Patients with blood eosinophil percentage ≥2% were defined as eosinophilic COPD patients. The cut‐off value for eosinophilic inflammation was defined as 2% because of reported high correlation rates with sputum eosinophilia2.5A detailed, structured questionnaire incorporating risk factors, smoking status, disease history, and comorbid conditions was completed for each patient. Exacerbation history was obtained from both medical records and the patients' declarations. LTOT use and domiciliary NIMV were recorded according to patient's declaration. Dyspnea severity was assessed using the modified Medical Research Council (mMRC) Dyspnea Scale and health‐related quality of life was assessed with St. George Respiratory Questionnaire (SGRQ).2.6t‐test, respectively. Candidate risk factors related with eosinophilic inflammation were evaluated firstly by univariate analysis and then possible risk factors with p values below 0.10 were evaluated by multiple logistic regression model to identify independent predictors of eosinophilic inflammation. Values of p < 0.05 were considered statistically significant.Data were analyzed using Statistical Package for Social Sciences (SPPS) version 22. Means and standard deviations were reported for normally distributed continuous data and medians and interquartile ranges (ICR) for nonnormally distributed continuous data. Exacerbation rates and hospitalization within previous year were reported according to one sample poisson distribution test. Correlations between nonnormally distributed data were calculated by the Spearman test. Comparison of the two groups with nonnormally and normally distributed data was analyzed with the Mann–Whitney U test and the Student 31 predicted % was 35.9 ± 11.3. By GOLD spirometric classification 86.6% of the patients were GOLD III or IV  and had higher FEV1 predicted % values (p = 0.03), Table p = 0.03) and less dyspnea with lower mMRC (p = 0.009) scores. We found that LTOT use was more frequent in patients with noneosinophilic inflammation (blood eosinophil <2%) compared with patients with eosinophilic inflammation (p = 0.01), Table Absolute number of blood eosinophil count and blood eosinophil percentage were 195 100–265] and 2.21% [1.3%–3.1%]. Eosinophilic inflammation (defined as blood eosinophil percentage ≥2%) was evident in 54 (60%) of the patients. Participants with eosinophilic inflammation were significantly older  and LTOT use .We included age, total number of exacerbations, mMRC score, SGRQ total score, FEVon Table . Eosinop41% predicted <50 or with ≥1 exacerbation leading to hospital admission or ≥2 moderate or severe exacerbation history. Eosinophilic COPD patients are older and have higher FEV1% predicted. These patients are characterized by fewer symptoms according to mMRC scores and better quality of life defined by SGRQ scores. Noneosinophilic COPD patients needed LTOT more frequently. Other clinical measurements like smoking status, comorbidities, and number of exacerbations in the last year were not significantly different between groups.The prevalence of eosinophilic inflammation defined as >2% eosinophils is 60% in our study population consisting of COPD patents with FEV1%, and lower SGRQ and mMRC scores.1%predicted, better BODE scores, and less symptoms measured by mMRC and SGRQ.1%predicted and higher rate of exacerbations in eosinophilic COPD patients.1%predicted did not differ between groups, lower rate of GOLD stage 1 was identified in eosinophilic group.1%predicted did not differ between eosinophilic and noneosinophilic patients.Determination of COPD disease severity is complex and includes lung functions, measure of breathlessness, quality of life, and exacerbation frequency. Our findings concerning age, lung functions, and severity of symptoms are in accordance with the study of Singh et al., which reported on 1483 patients from the ECLIPSE cohort. Singh et al. stated that patients with persistent eosinophil ≥2% were older, mostly male, with higher FEV1%predicted ≤50%). However, Vedel‐Krogh et al.In our study patients with noneosinophilic inflammation tended to have more frequent exacerbations leading to hospitalization and higher total number of total exacerbations. Lonergan et al. included 7220 COPD patient and had a mean 9‐year follow‐up period. The study showed that the rate of severe exacerbations was lower in high eosinophilic COPD patients, and patients with high blood neutrophil count were related with higher mortality.In our study, eosinophilic inflammation is inversely and independently associated with long‐term supplemental oxygen need. In studies evaluating eosinophilic exacerbations, there is no statistical difference between the two groups, regarding the need for long‐term supplemental oxygenWells et al. followed 678 moderate–severe COPD patients for 5 years to evaluate risk factors associated with the development of resting hypoxemia and supplemental oxygen need. This study showed a negative correlation between baseline emphysema and follow‐up oxygen saturation.4.11% predicted <50 or with ≥1 exacerbation leading to hospital admission or ≥2 moderate or severe exacerbation history which is a constraint on generalizations. There is also missing data regarding possible risk factors for long‐term supplemental oxygen need; known risk factors such as heart failure, emphysema, and pulmonary artery enlargementOur study population included only male patients with FEV51% predicted.Our study supports the growing evidence of eosinophilic inflammation phenotype in COPD with differentiable clinical characteristics. Eosinophilic inflammation is inversely associated with dyspnea severity measured by mMRC and LTOT use independently from age, total number of exacerbations, SGRQ total score, and FEVThe institutional ethical committee approved the study protocol (number: 2015‐17/21), and written informed consent was obtained from all participants.Nilüfer Aylin Acet Öztürk designed research/study, “performed research/study collected data, analyzed data, and wrote the paper. Aslı Görek Dilektaşli designed research/study, “performed research/study, collected data, analyzed data, and wrote the paper. Özge Aydin Güçlü designed research/study, “performed research/study collected data, and wrote the paper. Ezgi Demirdöğen designed research/study, contributed important reagents, analyzed data, and wrote the paper. Funda Coşkun designed research/study, contributed important reagents, analyzed data, and wrote the paper. Ahmet Ursavaş designed research/study, contributed important reagents, analyzed data, and wrote the paper. Mehmet Karadağ designed research/study, contributed important reagents, analyzed data, and wrote the paper. Esra Uzaslan designed research/study, contributed important reagents, collected data, and wrote the paper. All authors contributed in data collection and analysis. All authors have read and approved the final draft.Financial support was not provided.Authors have no conflict of interest regarding this paper.


Gastric cancer (GC) is the fifth most common malignant tumor and the third leading cause of cancer‐related deaths worldwide. CircRNAs may provide new insights into the development of GC by acting as oncogenes or tumor suppressors. In this study, we aim to examine the biological role of hsa_circ_0001944 (circFIRRE) in tumor progression of GC.The bioinformatic analysis, qPCR, Western blotting, and immunohistochemistry were fulfilled to detect the expression of hsa_circ_0001944, miR‐498, and GSPT1 in gastric cancer. Gain or loss of function approaches were used to investigate the biological functions of hsa_circ_0001944. MTS, EDU, wound healing, and transwell assays were performed to study the proliferation, invasion, and migration of GC cells. These molecular mechanisms were detected by luciferase reporter assays and chromatin immunoprecipitation assays.We screened out hsa_circ_0001944, whose expression was significantly increased in gastric cancer tissues. Knockdown of hsa_circ_0001944 significantly suppressed the cell proliferation, invasion, and migration. Mechanistic investigations showed that hsa_circ_0001944 can bind to and sponge miR‐498. Moreover, hsa_circ_0001944 sponged miR‐498 to increase GSPT1 expression, thereby promoted excessive proliferation and maintained the malignant phenotype of GC cells.The present study demonstrates the hsa_circ_0001944/miR‐498/GSPT1 axis contributes to GC development. This may provide a target for GC therapy and potential prognostic biomarker. We aim to examine the oncogenic role of CircFIRRE in gastric cancer. Knockdown of circFIRRE suppressed cell proliferation, invasion and migration. Moreover, circFIRRE sponged miR‐498 to increase GSPT1 expression, thereby promoted the malignant phenotype of gastric cancer. The present study demonstrates the circFIRRE/miR‐498/GSPT1 axis contributes to gastric cancer development. MiRNA sponging acts as the most widely reported mechanism of circRNAs' roles in developing cancers.G1 to S phase transition 1 (GSPT1) has been demonstrated oncogenic activity in gastric cancer.The current research observed that hsa_circ_0001944 was overexpressed in gastric cancer and can drive the malignant phenotype of gastric cancer cells. Hsa_circ_0001944 could regulate the expression of GSPT1 via miR‐498 sponging in gastric cancer. Therefore, our study found that the hsa_circ_0001944/miR‐498/GSPT1 axis can regulate the development of gastric cancer and may also give a hidden target for gastric cancer treatment.22.1Firstly, we analyzed the Gene Expression Omnibus (GEO) dataset GSE121445 and observed hsa_circ_0001944 was the highest up‐regulated circRNAs in gastric cancers compared with adjacent normal tissues Figure . Hsa_cir2.2Expression of hsa_circ_0001944 in different gastric cancer cell lines was evaluated by qPCR, AGS, and SGC7901 cell lines showed a relatively higher expression Figure . Thus, t2.3Since miRNA sponging is one of the leading mechanisms of circRNAs in the growth of several tumors, we furtherly predicted the potential target miRNAs of hsa_circ_0001944 based on CSCD and circInteractome databases. We observed that miR‐498 was the only candidate miRNAs for hsa_circ_0001944 in both CSCD and circInteractome Figure . The bin2.4All the MTS, EDU, transwell, and migration assays confirmed that knockdown of hsa_circ_0001944 inhibited the cell viability, propagation, aggression, and migration of SGC7901. However, these inhibiting effects were reversed after miR‐498 inhibition Figure . In conc2.5As described above, hsa_circ_0001944 could bind to and sponge miR‐498 in gastric cancer cells. Thus, according to TargetScan, miRDB, miRWalk, and Starbase databases, the bidding of miR‐498 to the 3′‐UTR of EP300, DHX35, KIF5B, LMAN1, TMEM184B, and GSPT1 Figure  could be2.6As reported above, hsa_circ_0001944 knockdown inhibited cell viability, proliferation, invasion, and migration of SGC7901. However, these inhibiting effects were reversed under GSPT1 overexpression Figure . Therefo2.7Thereafter, orthotopic xenografts were made to decide the role of hsa_circ_0001944 knockdown in vivo. Both tumor volumes and tumor weights were reduced after hsa_circ_0001944 knockdown Figure . Immunoh3Gastric cancer is one of the most life‐threatening malignancies worldwide.CircRNAs are closed‐loop RNAs abundant in various tissues of the human body and can be divided into noncoding and coding circRNAs.This research firstly confirmed increased expression of hsa_circ_0001944 in gastric cancer tissues. As reported, circ_0001944 contributed to glycolysis and tumor development by up‐regulating NFAT5 as a decoy for miR‐142‐5p in non‐small cell lung cancer.CircRNAs functions in various mechanisms, such as RBP sponge, miRNA sponge, directly regulating mRNA transcription, or translation. For instance, the expression of circRNA_100269 is reduced in gastric cancer, and it can suppress the development of tumor cells by targeting miR‐630 (31). MiRNA sponging is one of the most important molecular mechanisms of circRNAs. This research proved that hsa_circ_0001944 could bind to and sponge miR‐498 in gastric cancer cells, and the inhibiting effects of hsa_circ_0001944 knockdown could all be reversed by miR‐498 inhibitor treatment.As previously described, circRNAs can regulate mRNA expression via miRNA sponging. Our study forecasted that miR‐498 could bind to the 3′‐UTR of GSPT1. GSPT1 acts as an oncogene in gastric cancer. Therefore, GSPT1 could be a reasonable downstream gene of hsa_circ_0001944 to exert its cancer‐promoting effect. Meanwhile, we confirmed that hsa_circ_0001944 sponged miR‐498 and subsequently regulated GSPT1 expression in gastric cancer cells. GSPT1 overexpression also reversed the inhibiting effects of hsa_circ_0001944 knockdown. Meanwhile, the same conclusion in vivo was reached. Therefore, our research demonstrated that hsa_circ_0001944 enhanced GSPT1 expression via sponging miR‐498 to promote gastric cancer cell proliferation and invasion.The current research demonstrates the circFIRRE/miR‐498/GSPT1 axis contributes to gastric cancer development, which may offer a target for gastric cancer therapy and potential prognostic biomarker.44.1Shanghai Cancer Center, Fudan University offered forty paired samples from gastric cancer patients and the nearby normal tissues. The detailed clinical information was listed in Table 4.22.Offered by the Chinese Academy of Sciences cell bank, the human gastric cancer cell line AGS and SGC7901 were cultured in RPMI1640 , added with 1% penicillin/streptomycin (Gibco), and 10% fetal bovine serum (FBS) at 37°C with 5% CO4.3The lentivirus transfection and effect identifications were made based on the manufacturer's guidance. Gene‐Chem was adopted for constructing the vectors based on lentivirus for the overexpression of hsa_circ_0001944 and GSPT1, RNAi‐regulated knockdown of hsa_circ_0001944, and their negative regulation. The miR‐498 inhibitor , mimic , and Thermo Fisher Scientific (Assay ID: 4464076) offered the negative controls. qPCR was used to detect the transfection efficacy. Table 4.4The qPCR was made based on the manufacturer's guidance. To be brief, the total RNA of gastric cancer tissues and cell lines was extracted by the Mini‐BEST Universal RNA Extraction kit (TaKaRa). In terms of circRNA and mRNA, the reserve transcription of RNA was made into cDNA via the Prime Script RT Master Mix reagent kit (TaKaRa). With regard to miRNA, the Prime Script™ RT reagent kit (TaKaRa) was adopted for synthesizing cDNA. Then, the SYBR Green Master Mix (TaKaRa) with PCR LightCycler480 (Roche Diagnostics) was employed to make the qPCR assays. The TaqMan Universal Master Mix II (Applied Biosystems) was adopted for detecting the expression of miR‐498. Thermo Fisher Scientific (Primer ID: Hs00502366_CE) offered the primers of miR‐498. Table 4.5A total cell protein extraction kit (KeyGen Biotechnology) was adopted for separating total proteins of gastric cancer tissues or cells. Next, following SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE), the transfer of total protein for every sample onto the polyvinylidene difluoride (PVDF) membranes was conducted. Next, 2% bovine serum albumin (KeyGen Biotechnology) was adopted to block PVDF membranes at room temperature for 2 h, and the overnight incubation of all these membranes was made at 4°C with the primary antibodies below: GSPT1  and β‐actin . After 2 h of secondary antibody incubation, the detection of all the bands was made with a chemiluminescence ECL kit (Beyotime Biotechnology), and all the bands were quantified with the ImageJ software . The calculation of the expression of proteins was made on basis of the internal regulation β‐actin.4.6Embedded in paraffin, gastric cancer tissues were sliced into 4 mm sections and labeled with primary antibodies as follows: Ki67 , GSPT1 . Then, after the treatment with an immunohistochemical labeling kit (MaxVision Biotechnology), the slices were imaged via a light microscope (Olympus). According to the German immunohistochemical mark, the expression degrees and the staining intensity were evaluated.4.74 cells/well, the transfection of gastric cancer cell line AGS and SGC7901 with relative luciferase reporter plasmids was made, followed by 48‐hour relative treatment. In the end, relative luciferase activities were evaluated via the Dual‐Luciferase Reporter Assay System (Promega) and calculated as the proportion of firefly luciferase activity to Renilla luciferase activity.Luciferase reporter assays were made based on the manufacturer's instructions. Briefly, Gene‐Chem was adopted for constructing all the luciferase reporter plasmids . Then, after being seeded into 96‐well plates at a density of 1 × 104.8Based on the producer's guidance, the RIP assays were made via an Imprint RNA Immunoprecipitation Kit (Sigma). Firstly, the lysis of all gastric cancer cell line AGS and SGC7901 under the varied conditions was made in RIP buffer, such as magnetic beads conjugated with the negative control IgG and anti‐AGO2 (Millipore). Then, the immunoprecipitated RNAs were obtained after incubation with Proteinase K buffer (Omega). In the end, the detection of precipitants was made with the qPCR assays.4.93 cells/well, followed by incubation for 0, 24, 48, 72, 96, and 120 h. Then, according to the manufacturer's guidance, the detection of cell viability under different conditions was made by the CellTiter 96® Aqueous Non‐Radioactive Cell Proliferation Assay Kit (Promega).Cell viability assays were made based on the manufacturer's guidance. Briefly, the seeding of gastric cancer cell line AGS and SGC7901 into 96‐well plates was made at a density of 1 × 104.105 cells/well for 24 h. Next, 10 μM EDU reagent was put to the medium, followed by 2‐hour incubation at 37°C with 5% CO2. After fixture with 4% paraformaldehyde (solarbio) and permeabilization with 0.5% Triton ×‐100 (solarbio), the AGS, and SGC cells were counterstained. In the end, a laser scanning confocal microscope (Olympus) was adopted to evaluate the percentage of EDU‐positive cells.EDU assays were made based on the producer's guidance. Briefly, the EDU assays were adopted for detecting the proliferation of gastric cancer cell line AGS and SGC7901 via the EDU assay kit  based on the producer's guidance. Firstly, the seeding of AGS and SGC7901 cells into 24‐well plates was made at 1 × 104.114 cells per mouse) was made. The daily observation of every group was conducted for no distress or death sign, followed by sacrificed mice and isolated whole tumor. Finally, tumor volume and tumor weight were calculated. A Vernier caliper detected the tumor size, and the formula below: V = (D × d)/2 mm was adopted to calculate the tumor volume, where D means the longest diameter and d refers to the shortest diameter of the tumor.Based on the Animal Care Committee of Shanghai Cancer Center, Fudan University, xenograft experiments were made as illustrated.4.12http://www.circbase.org/). Cancer‐Specific circRNA database  and CircInteractome (https://circinteractome.nia.nih.gov) databases were adopted for predicting the sponging miRNAs of circFIRRE. MiRDB (http://mirdb.org), TargetScan (www. targetscan.org), miRWalk , and Starbase (http://starbase.sysu.edu.cn) were adopted for predicting possible the targeting mRNAs of miR‐498.The basic information of hsa_circ_0001944 was acquired from circBase  was adopted to make the statistical analysis. The two‐tailed Student's This work was supported by the National Natural Science Foundation of China 82203752.The authors disclose no potential conflicts of interest.Figure S1.Click here for additional data file.Table S1.Click here for additional data file.Table S2.Click here for additional data file.Table S3.Click here for additional data file.