Bacterial vaginosis (BV) increases HIV risk and adverse reproductive outcomes. Standard-of-care (SOC) for BV are antibiotics; however, cure rates are low. Probiotics for vaginal health may be useful in improving cure and recurrence although the regulatory framework governing probiotics and the conduct of randomized clinical trials to evaluate these has not been established in South Africa. We performed an exploratory single-blind trial evaluating a commercial oral-vaginal-combination probiotic as adjunct to SOC for BV treatment.n = 12) or to metronidazole followed by a commercial oral/vaginal probiotic (n = 18). The primary qualitative outcome was to test the regulatory landscape for conducting randomized probiotic trials in South Africa; and acceptability of vaginal application by women. BV cure at 1 month (Nugent≤3) was the primary quantitative endpoint. Secondary quantitative endpoints were BV recurrence, symptoms, vaginal microbiota and genital cytokine changes over 5 months post-treatment.Women with symptomatic vaginal discharge were screened for BV and common sexually transmitted infections (STIs). BV+ (Nugent 7–10) but STI- women were randomized to vaginal metronidazole alone  reviewed and approved this trial. As probiotics continue to be regulated as health supplements in South Africa, SAHPRA required a notification application for this trial. Acceptability and adherence to the oral and vaginal application of the probiotic were high, although women reported a preference for oral capsules. 44.8% of women cleared BV one-month post-treatment, and no significant differences in BV cure , recurrence, vaginal pH, symptoms, microbiota or vaginal IL-1α concentrations were found between SOC and intervention groups in this pilot study with an over-the-counter product.Navigation of the SAHPRA registration process for evaluating a commercial probiotic in a randomised trial laid the foundation for planned larger trials of improved probiotic products for vaginal health in South Africa. Although adherence to the vaginally delivered probiotic was high, women preferred oral application and we recommend that improvements in the content and method of application for future probiotics for vaginal health should be considered.http://www.sanctr.gov.za/; BV-trial1; DOH-27-1117-5579).This trial was registered on 17 October 2017 with the South African National Clinical Trial Register ( Lactobacillus-dominated to a polymicrobial microbiota, compromising a mixture of diverse anaerobes, such as Prevotella, Gardnerella, BVAB1, Sneathia, and Megasphera spp. [Bacterial vaginosis (BV) is the most common genital condition of women of reproductive age , definedera spp. –4. It inera spp. , 6 and aera spp. , 8, possera spp. . Antibioera spp. , 11. Whiera spp. , more thera spp. . In Afriera spp.  and geniera spp. , an urgeLactobacillus-containing probiotics as an adjunct to antibiotics in treating BV [Several clinical studies have evaluated ating BV , 17. Howating BV . As the ating BV –21, it iating BV , 23, onlating BV .In South Africa, the registration of medicines, probiotics and health supplements along with their use in clinical trials is regulated by the South African Health Products Regulatory Authority (SAHPRA), but none of the > 100 currently available over-the-counter (OTC) probiotics  has prevThis trial was submitted and approved by SAHPRA , the University of Cape Town’s (UCT) Human Research Ethics Committee (HREC Ref 706/2016) and the South African National Health Research Ethics Council (NHREC Ref 4579) and registered with the South African National Clinical Trials Register of the Department of Health (DOH-27-1117-5579).C. trachomatis, N. gonorrhoea, T. vaginalis, and M. genitalium by TaqMan® Assays (Fast Track Diagnostics). Inclusion criteria were being BV positive (Nugent 7–10) but negative for any STI. Exclusion criteria included being pregnant, breastfeeding, pelvic inflammatory disease, living with HIV, having a known allergy to metronidazole, and/or currently using any other antibiotics or natural remedies in the urogenital area. Women who acquired an STI or had recurrent BV over the course of the trial were referred for treatment but not excluded, and any concomitant medication (including antibiotics) taken over the course of the study was recorded. Study visits were planned such that women were not menstruating nor reported having unprotected sex or douching in the 48 h prior to sampling. All women were tested for HIV (Rapid Anti-HIV (1&2) test; InTec products, Inc., China) and pregnancy  at screening.Women were recruited from a South African public sector STI clinic (Spencer Road Clinic) and from the UCT Student Wellness Centre in Cape Town, South Africa. Eligible women were 18–45 years old and seeking care for vaginal discharge. All eligible women were tested for BV (by Nugent scoring) and STIs, including 9 CFU of L.casei rhamnosus for 7 days and Anukam et al. (2006) reported cure rates of 88% in the treatment group and 40% in the control group after administering 500 mg metronidazole orally for 7 days followed by oral administration of L.rhamnosus and L.reuteri (each 109 CFU) for 30 days. In both studies, cure rates were assessed 30 days after the last administration of the probiotics. Since we applied probiotics both orally and vaginally and for 15 days, we expected the cure rates to be similar. Thus, we anticipated a sample size of 30 women (enrolled in a 2:3 ratio in SOC: intervention arms) would detect the above difference in proportions (0.83 vs. 0.35) with 75% power at α = 0.05, which we considered sufficient for this pilot trial.This was an exploratory study designed to establish the regulatory landscape to conduct probiotic trials in South Africa, with additional exploratory quantitative analyses. We based our sample size calculation of 30 women on cure rates from previous studies , 26. PetRandomisation was performed using the pseudorandom number generator in Microsoft Excel 2016 (MT19937) by research pharmacists at the UCT CRC who were not involved with clinical procedures and/or screening processes and only dispensed the investigational product. Researchers and laboratory staff involved in sample and data analysis were blinded to the randomisation process. The research nurse who conducted the clinic visits was not blinded as she interacted directly with participants. After the database lock and a primary blinded analysis, the unblinded treatment allocations were released.L. acidophilus, L. rhamnosus GG, B. bifidum and B. longum at ≥2 × 109 colony-forming units (CFU).Eligible women either received topical metronidazole only  or topical metronidazole followed by a 15-day treatment course of a probiotic marketed in South Africa to improve vaginal health (intervention group), which included 5 days of oral probiotic capsules followed by 10 days of oral capsules together with twice daily vaginal spray. The oral capsules and each metered dose of the vaginal spray contained lyophilized The contents and concentrations of each microbial species of the probiotic lot were confirmed prior to initiation of the trial , and 1, 3 and 5 months post-treatment, genital samples were collected in the following order under speculum examination: (1) a vulvo-vaginal swab for STI testing; (2) a posterior fornix and lateral wall swab to screen for BV (by Nugent Scoring) and to measure vaginal pH ; and  two lateral wall swabs to measure genital IL-1α  and vaginal microbiota (by qPCR). In addition, women completed a questionnaire on demographics, reproductive health and sexual behaviour at enrolment, and a questionnaire assessing feasibility, acceptability and adherence to the administered products, vaginal symptoms and adverse events at follow up visits. Product preference was assessed at the final visit by questionnaire , C. trachomatis (ATCC® VR-885), T. vaginalis (ATCC® 30001) and M. genitalium (ATCC® 33530). BV was diagnosed by Gram staining of vaginal smears and Nugent Scoring. Slides were assessed microscopically and assigned a score between 0 and 10, with a score of 0–3 considered BV negative, 4–6 intermediate microbiota, and 7–10 BV positive.The commercial TaqMan® FTD STD9  kit, performed as per manufacturer’s instructions, was used to test for L. acidophilus, L. rhamnosus, B. bifidum and B. longum) and bacterial species associated with vaginal health  or BV , and IL-1α concentrations  over 5 months post-treatment.While this study was intended to test the regulatory landscape to conduct probiotic trials in South Africa and acceptability/preference of the probiotic administration for vaginal health, BV cure rates were considered (defined as having Nugent≤3 one-month post-treatment) as primary quantitative endpoint. In addition, other secondary endpoints included adherence, recurrence, changes in vaginal pH, concentrations of bacterial species contained in the administered probiotic , BV-associated organisms , and bacterial species contained in the administered probiotic product  were assessed using commercially available Applied Biosystems™ TaqMan® Assays . For L. vaginalis (KF875988.1) and L. mucoasae (NR_024994.1) Thermo Fisher Scientific designed custom probes based on the referenced nucleotide sequence. The TaqMan probe sequences for L. acidophilus and L. rhamnosus  were prSamples were thawed on ice and filtered by centrifuging at 1950 g for 10 min at 4 °C in SPIN-X® 0.2 μM cellulose acetate filters to exclude mucus and debris prior to performing the ELISA. Human IL-1α concentrations were measured using a commercial ELISA , according to the manufacturer’s instructions. IL-1α concentrations were calculated based on the standard curve, using standards provided with the kit. The detection range was 1.25–125 pg/mL, and all values below the detection limit were recorded as half of the lowest concentration measured.p-values ≤0.05 were used to determine statistical significance.GraphPad Prism6® , STATA version 11.0  and R were used for descriptive and statistical analyses and to generate graphs. Planned description of continuous variables with means, medians and standard deviations, as appropriate, were calculated. Categorical variables were described as proportions. Mann-Whitney U tests were used to compare groups of continuous variables and relative risk (RR) was used for categorical variables. 95% confidence intervals and This trial intended to explore the regulatory environment in South Africa governing probiotic trials, in order to lay the foundation for future trials of novel probiotic products. Prior to conducting this randomized trial, no probiotic trial had been approved by the regulatory authorities in South Africa. Thus, discussions with SAHPRA authorities were initiated in March 2016, as a decision on whether a full application for the trial was necessary, given that this was a randomized trial with a vaginally applied product, or whether a notification application was required. Currently, already available OTC probiotics  are not considered or regulated as a medicine in South Africa but rather as a health supplement. Typically, the regulator requires full applications for products not registered in South Africa or for those not being used for their registered indication, dose, or formulation. Alternatively, SAHPRA notification is required for phase IV clinical studies of an approved medication within its approved dosage, formulation and indication. SAHPRA finally confirmed that the trial did not require a full application but rather a notification application, which was submitted to SAHPRA and approved .n = 90) were recruited via the UCT Student Wellness Centre. One woman tested HIV positive at screening, and thus was excluded and referred for care. Of 95 women who were eligible for screening, 43 women (45.3%) were confirmed to have BV  and C. trachomatis  being the most common STIs detected. Of the 43 BV positive women, 33 were eligible for randomization (BV+ but STI-).Between 30 October 2017 and 22 March 2018, a total of 96 women seeking care for vaginal discharge were screened for eligibility  reported previously being diagnosed or treated for an STI , while 18/30 were randomized to the intervention arm . Randomised women were a median age of 22 years (IQR 20–26 years) old, predominantly single , and self-identified as black . Their median age at sexual debut was reported to be 17 years (IQR 16–19 years) with a median of five lifetime sexual partners (IQR 3–8), and reporting of oral sex was common . About half (18/30) reported regular condom use, and few  completing all visits , flu-like symptoms (n = 18), STI acquisition, including C. trachomatis (n = 8), T. vaginalis (n = 7), M. genitalium (n = 5) and N. gonorrhoea (n = 1), menstrual pain (n = 7), gastro-intestinal complaints (n = 5), temporary vaginal discomfort related to vaginal intercourse (n = 7), anaemia (n = 2), cat allergy (n = 1), buttock pain (n = 1), and anxiety (n = 1). It was concerning that more than half of the women  acquired an STI over the course of the study, supporting that BV is considered a risk factor for acquisition of STIs [Overall, a total of 110 adverse events (AEs) were recorded over the course of the study. Most were mild  and unrelated to study product use 76.4%; 84/110), including headaches , the majority was associated with vaginal metronidazole use and included previously describe side effects of vaginal metronidazole gel [Of AEs considered to be related to study products  reported that they liked using the combination probiotic because it was easy to use or resulted in improved vaginal symptoms Fig. a. Most wThis trial was intended as an exploratory study to test the local regulatory environment and acceptability of vaginal probiotics. However, we also conducted an exploratory intention-to-treat (ITT) analysis to estimate possible benefits of administration of this locally sourced probiotic on clinical outcomes. The majority of women reported an improvement of their vaginal symptoms post-treatment, including discharge, colour and odour, with similar rates in the SOC and intervention arms Table . Of thesp = 0.109) 1 month post-treatment  was 44.8% at month one, 46.4% at month three and 53.8% at month five. BV cure rates tended to be higher in the SOC arm  compared to the intervention arm .In the SOC arm, 6/11 (54.5%) women had a vaginal pH < 4.5 1 month after treatment, compared to 11/18 (61.1%) in the intervention group , B. bifidum (2/30) and B. longum (8/30). However, we found no evidence of colonization of any of the bacterial species contained in the probiotic formulation post-treatment , P. bivia (21/30) and A. vaginae (29/30) pre-treatment  present pre-treatment, only some women lost or had decreased quantities of these bacterial species post-treatment, irrespective of study arm , and 3 months post-treatment .Genital IL-1α, a marker of inflammation , decreasIn the present study, we evaluated the regulatory environment for conducting probiotic trials in South Africa. SAHPRA required a notification application, as the probiotic was available OTC in South Africa and considered a health supplement. The use of a vaginal/oral probiotic combination was well accepted among South African women included in this exploratory study and was associated with few product-related AEs. While this study was not sufficiently powered to test efficacy of this convenient OTC probiotic in South African women, we observed no beneficial clinical effect of this adjunctive probiotic compared to vaginal metronidazole alone as SOC. Nonetheless, this exploratory trial provided valuable lessons for future probiotic trials of the same disease entity in the same population, with improved products and adequate sample sizes.With regards to the regulatory landscape, SAHPRA regulates OTC probiotics currently as health supplements, independently of their route of administration and health claims being made, but labels must state that “This unregistered medicine has not been evaluated by the SAHPRA for its quality, safety or intended use” . In contLactobacillus spp. should be administered orally. If topical administration is required, a vaginal tablet or gel would be preferred over a spray. Importantly, women were not willing to spend more than ZAR 200 (~USD11.40) for one treatment course. Given that > 60% of South African households earned less than ZAR 6367 (~USD368.55) per month according to the latest Census [With regards to product design, women reportedly preferred the oral over the vaginal application of the probiotic, as the administration of the vaginal spray was uncomfortable and messy. Thus, for future probiotic product design women’s preferences should be taken into consideration and probiotics containing well-selected vaginal t Census , the cosLactobacillus spp. that are commonly present in the FGT and associated with vaginal health, internationally and in South African women, such as L. crispatus, L. gasseri or L. jensenii [Lactobacillus spp. are highly adapted to this ecological niche [Lactobacillus spp., they might have failed to colonize, which is in agreement with our microbiological results. For a successful colonization and subsequent ability to confer a health benefit to the host, the bacteria need to fulfil a broad range of other criteria including adherence to vaginal epithelial cells, production of lactic acid, decrease of vaginal pH, decrease of inflammatory cytokines, and inhibition of BV-associated bacteria, such as G. vaginalis or P. bivia [Lactobacillus strains contained in this OTC phenotypically and compared their characteristics to vaginal Lactobacillus strains from healthy South African women [Lactobacillus strains largely performed better in these in vitro assays than the strains currently used in probiotics for vaginal health, suggesting that it might not be ideal to include these strains in commercial probiotics for vaginal health.The selected OTC oral/vaginal probiotic, like all those available in South Africa, does not contain jensenii , 43. Othal niche , and sinBased on this experience with the South African regulatory body SAHPRA, we have some important insights for the design of future probiotic trials in South Africa. Clinical studies should test for BV cure shortly after treatment completion to minimize the likelihood that women have a recurrent BV event, in order to differentiate BV cure rates from confounding repeat episodes. Further, including a comparison with vaginal metronidazole gel alone or in combination with probiotic is more similar to a superiority trial rather than placebo-controlled , which iL. iners correlated positively with the inflammatory cytokine IL-1α. This supports literature suggesting that L. iners is not necessarily as beneficial as other common vaginal Lactobacillus spp. [L. iners is often present during BV, has complex nutritional requirements compared to other vaginal Lactobacillus spp., and encodes inerolysin, a pore-forming toxin that is related to G. vaginalis-encoded vaginolysin [L. iners in our study suggests that rather than only treating women with BV, also women with an L. iners-dominated microbiota and high genital inflammation could benefit from well-designed probiotics for vaginal health.One interesting microbiological observation made during this study was that concentrations of vaginal lus spp. –50. L. iAn important limitation of this study was the small number of participants; although this was intended as an exploratory study to test the regulatory environment in South Africa and the acceptability/preference of oral versus topical application. In addition, administration of the study drugs was self-reported and vaginal pH was not being measured pre-treatment. It was also unexpected that genital tract IL-1α levels differed at baseline between randomized groups, which is difficult to explain. Further, no 16S rRNA gene sequencing to evaluate relative vaginal bacterial abundance was performed, neither was bacterial culture.Lactobacillus strains are needed to conclusively determine the efficacy of adjunctive probiotics on BV cure and recurrence in South African women.In a region with some of the highest rates of BV, STIs and HIV, it is critical that the local regulatory environment for conducting randomized trials of planned novel probiotic products is established. This randomized single-blinded trial provided important regulatory and clinical considerations critical in the design of future clinical trials, with larger sample sizes intended for safety and efficacy endpoints. This exploratory trial did suggest that oral and vaginal OTC probiotics were generally safe and acceptable in South African women with BV. Due to the critical need to manage BV better internationally and in South Africa, future double-blind, randomized, placebo-controlled trials, using a probiotic product containing beneficial, well-characterised vaginal Additional file 1. Participant questionnaires. Questionnaires administered to the participants to capture data on demographics, women’s reproductive and sexual health, use and preference of study products.Additional file 2: Figure S1. Bacterial concentration per dose unit of the commercial probiotic. The CFU per dose of three boxes  of the probiotic lot containing oral capsules (C) and vaginal spray (S) was determined using serial dilutions and compared to the manufacturers claim of 2 × 109 CFU per dose (dotted line).


Although abnormal mitosis with disarranged metaphase chromosomes or many micronuclei in astrocytes (named “Alzheimer I type astrocytes” and later “Creutzfeldt-Peters cells”) have been known for nearly 100 years, the origin and mechanisms of this pathology remain elusive. In experimental brain insults in rats, we show that abnormal mitoses that are not followed by cytokinesis are typical for reactive astrocytes. The pathology originates due to the inability of the cells to form normal mitotic spindles with subsequent metaphase chromosome congression, which, in turn may be due to shape constraints aggravated by cellular enlargement and to the accumulation of large amounts of cytosolic proteins. Many astrocytes escape from arrested mitosis by producing micronuclei. These polyploid astrocytes can survive for long periods of time and enter into new cell cycles. Here weAdult male rats were housed in standard cages with free access to food and water on a 12-h light/dark cycle. All procedures performed on animals were approved by Columbia University’s Institutional Animal Care and Use Committee and conducted according to institutional and federal guidelines.After premedication with scopolamine  to prevent the effects of peripheral cholinergic stimulation, pilocarpine  was administered to Sprague-Dawley rats (100–150 g) to induce seizures. Seizures were graded on the modified Racine scale , and onlKainic acid dissolved in isotonic saline (pH 7.4) was given i.p. to Sprague-Dawley rats (100–150 g) at 10 mg/kg with repeated injections of the same dose over 30 min until the appearance of grade 4–5 seizures, according to the modified Racine scale. After 2 h of continuous seizures, ketamine  was administered to stop seizures, and a second dose  was administered if seizures did not stop in 10 min after the first.Sprague-Dawley Rats (100–250 g) were anesthetized  and placed in a stereotactic frame and the skull was exposed using sterile technique. After drilling the skull, a blunt 26-G needle (Hamilton) was inserted into the frontal cortex. 10 μl of solution  was administered. After 96 h, animals were deeply anesthetized with an overdose of ketamine/xylazine, and perfused with 4% paraformaldehyde (PFA).Wistar rats (275–300 g) were subjected to transient middle cerebral artery occlusion using a method of intraluminal vascular occlusion . The ani0 C). 40 μm sections were prepared with a vibratome (Leica VT1000S) and stored in cryoprotectant solution at − 200 C. Standard procedure for Nissl staining with Cresyl violet was used for routine analysis of tissue.After perfusion brains were removed and additionally fixed in 4% PFA in PBS for 14–18 h (4Primary antibodies: (1) markers of astrocytes: (i) glial fibrillary acidic protein (GFAP): mouse monoclonal , rabbit polyclonal , phospho-GFAP (Ser8) mouse monoclonal ; (ii) vimentin: monoclonal , phospho-vimentin (Ser55): mouse monoclonal ; (iii) nestin: rabbit polyclonal ; astrocyte specific glutamate transporters: (iv) GLAST: monoclonal ; (v) GLT1: mouse monoclonal ; (vi) calcium binding protein specific for glial cells - S100: rabbit polyclonal ; (vii) alpha-B crystallin: rabbit polyclonal ; (2) marker of NG2 cells: NG2 Chondroitin Sulfate Proteoglycan ; (3) marker of microglial cells: Iba1, rabbit polyclonal ; (4) markers of DNA damage: phospho-gamma-H2AX (Ser139), mouse monoclonal ; (5) chromosome markers in dividing cells: (i) Ki67: mouse monoclonal  and rabbit polyclonal ; (ii) PCNA: mouse monoclonal ; (ii) phospho-Histone H3(Ser10): rabbit polyclonal  and phospho-Histone H3 (Ser10): mouse monoclonal ; (6) centrosome markers, (i) pericentrin: rabbit polyclonal ; (ii) gamma-tubulin: mouse monoclonal  and goat polyclonal ; (7) markers of microtubules: (i) alpha-tubulin: mouse monoclonal ; (ii) TPX2: rabbit polyclonal ; (8) marker of nuclear envelope: Lamin A/C: rabbit polyclonal : (9) markers of proteins activated in mitosis: (i) Aurora A: mouse monoclonal  and rabbit polyclonal ; (ii) Aurora B: rabbit polyclonal ; (iii) Bub3: mouse monoclonal  and rabbit polyclonal ; (iv) BubR1: mouse monoclonal  rabbit polyclonal ; (v) NUMA: rabbit polyclonal ; (vi) Survivin: rabbit polyclonal ; (10) Lucifer yellow: rabbit polyclonal .Secondary antibodies conjugated to fluorophores: anti-mouse Alexa Fluor 488, 594, and 633, anti-rabbit Alexa Fluor 488, 594, and anti-goat Alexa Fluor 488, 594, 633; all from goat or donkey .0 C). Alexa Fluor -conjugated secondary antibodies were used for 1 h at RT. For visualization of nuclei and chromosomes fluorescent Nissl reagent  and DAPI  were applied with secondary antibodies.For double- and triple-immunofluorescence, after blocking with 10% normal goat (or donkey) serum , free-floating sections were incubated in a mixture of primary antibodies raised in different species overnight . To control for the specificity of immunostaining, primary antibodies were omitted and substituted with appropriate normal serum.Slides were viewed using a confocal microscope (Nikon Ti Eclipse). 3D reconstructions were done from stacks of images with confocal microscope software NIS-Elements.5-Bromo-2′-deoxyuridine  was dissolved in sterile DPBS (10 mg/ml) and given i.p. at 80 mg/kg. At different time-points (see Results) animals were deeply anesthetized with an overdose of ketamine/xylazine, and perfused with 4% PFA. Vibratome sections were used for immunohistochemical detection of BrdU with anti-BrdU mouse monoclonal  and rat monoclonal  antibodies after DNA denaturation: 30 min treatment with 2 M HCl (RT) followed with 20 min treatment with 0.1 M sodium borate buffer (pH 8.5) at RT.TUNEL was performed with DeadEnd™Fluorometric TUNEL system  according to the manufacturer’s recommendations on vibratome sections. Regular procedures for immunohistochemical staining was performed after TUNEL.For regular transmission EM, animals were deeply anesthetized with a ketamine/xylazine, and perfused with 2% paraformaldehyde and 2.5% glutaraldehyde in PBS. After postfixation in 2% osmium tetroxide in 0.2 PB (2 h at 4 °C) and dehydration, pieces of tissue were embedded in Epon-Araldite . Ultrathin sections were cut with a Reichert Ultracut E, stained with uranyl acetate and lead citrate, and examined with a JEOL 1200 electron microscope.2, 1.5 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 Dextrose. The slices recovered at 30 °C in a chamber for at least 1 h before electrophysiological recording. The above solution was also used for whole-cell patch-clamp recording in brain slices at 30 °C. The bath solution was applied at a flow rate of 1.5 ml/min using the VC-6 perfusion valve control system (Warner Instruments) with the TC-344B temperature controller. Cells were visualized under a LEICA DMLFS microscope with a 63× water-immersion lens. The intracellular solution in the patch pipettes contained the following (in mM): 140 KCl, 1 MgCl2, 10 EGTA, 10 HEPES, 3 MgATP, 0.3 Na2ATP, pH 7.3 with KOH. Pipette resistance was ∼3–5 MΩ. Cell capacitance and series resistance were measured using the software MultiClamp 700A Commander Ver. 1.1.2.27 and pClamp 8 . Cells were initially identified morphologically based on the sizes and shapes of their somas and the architecture of their processes. For analysis of cell morphology and gap junction coupling with the surrounding cells, Lucifer yellow  was added to the intracellular solution  and filtered through a 0.2 μm PTFE filter. After the experiment, slices were fixed in 4% paraformaldehyde in PBS overnight at 4 °C. Slices were immunostained, and observed under a confocal microscope, as above.Coronal slices of rat forebrain (160–180 μm) were cut with a Vibratome (LEICA VT 1000S) in ice-cold oxygenated-modified artificial CSF (aCSF) (in mM): 125 NaCl, 2.5 KCl, 2 CaClThe numbers of mitotic astrocytes visualized with Ki67 or BrdU were counted in the images  obtained from neocortices in coronal sections .Levels of GFAP immunofluorescence were evaluated in the images obtained as described above from coronal brain sections stained for GFAP and Ki67. Images were transferred to Image J 1.46r (public domain), grayscaled and quantified based on the optical density (OD).t-test and one-way Anova. p < 0.05 was considered significant.Data are expressed as mean ± SEM. Continuous parameters were analyzed with Student’s We found many abnormal mitoses in astrocytes in excitotoxic (kainic acid and pilocarpine-induced), ischemic, and traumatic (stab wound) models of brain injury in rats Fig.a. These To determine whether abnormal mitoses might be due to DNA damage, we used TUNEL (as a marker of DNA cleavage) and phospho-gamma-H2AX  to evaluate DNA damage in astrocytes. Only one TUNEL+ astrocyte in C-mitosis was found out of 376 cells detected in C-mitosis and with many small nuclei on days 3–6 after induced seizures , whereas others showed ball-like cell bodies with only a few main processes, as judged by GFAP and vimentin immunostaining  were often displaced into the proximal parts of the processes at distances up to 11 μm from the spindle poles (defined as a point at which the kinetochore microtubules converged)  , 18. In SAC prevents the onset of anaphase before all chromosomes are properly attached to kinetochore microtubules and therefore can be segregated correctly , 30, 38.In arrested mitoses the distribution of BubR1 and BUB3 was altered. Their immunoreactivity predominated in kinetochores of chromosomes that were located apart from the main group of chromosomes Fig. g,h. WhenAurora B kinase is responsible for the attachment of kinetochore microtubules to chromosomes and fixes incorrect connections between chromosomes and microtubules , 28. It 2 in severe vs 0.5 ± 0.3 per 1mm2 in mild astrogliosis, P < 0.001).After the breakdown of the nuclear envelope, the cytoplasm gains access to the peri-chromosomal space and may interfere with chromosome congression. In normal mitosis, the area occupied by the mitotic spindle and chromosomes is free from many cytoplasmic proteins, for example, GFAP, the major intermediate filament of astrocytes Fig. a,b. HoweExamination of the ultrastructure of arrested mitoses showed that intermediate filaments were undetectable in the cell body around chromosomes Fig.a,b, wherAnother protein that accumulates in large amounts is the molecular chaperone, alphaB-crystallin. Immunostaining of astrocytes in abnormal mitoses revealed a diffuse cytoplasmic distribution of immunolabeling and alphaB-crystallin surrounded many of the chromosomes Fig. e.Reactive astrocytes escaped arrest in metaphase with the formation of many small nuclei. The appearance of multinucleated cells in cell culture has been usually considered as evidence of mitotic catastrophe and cellular demise , 51, butWe observed several months after pilocarpine-induced seizures that multinucleated astrocytes could enter the cell cycle and divide Fig. d-i. SeveNeuropathologists have recognized “Alzheimer Type 1 astrocytes”/“Creutzfeldt cells” for decades , 12, remThe appearance of Creutzfeldt cells varies in different pathological injuries and between species. For example, in pilocarpine-induced seizures in mice there are fewer of these abnormal astrocytes than in rats (unpublished data). Although we found Creutfeldt astrocytes in all models of injury in the rat brain, pilocarpine seizures produced the largest number. It is difficult to draw comparisons with human CNS injury, but as a general statement, Creutzfeldt astrocytes are described in a number of human neuropathological states but appear to be fewer in number than in the model we have chosen to examine. However, the human Creutzfeldt astrocytes appear to have many similar, even identical, pathological characteristics as the ones we illustrate in our study.We observed marked changes in astrocyte shape, including the loss of fine processes, partial retraction of main branches and the rounding up of cell bodies when we examined mitotic, hypertrophic astrocytes. The phenomenon of cell rounding during mitosis has been described and characterized in most detail in cultured cells. Here, typically over a period of 5–20 min, flat cells take on a near-spherical shape . Such shAstrocytes, in contrast, are process-bearing cells with complex geometry. Astrocytes divide in the young mouse brain without losing miniature peripheral processes . SimilaDuring a normal mitosis, centrosomes are attached to kinetochore, polar, and astral microtubules, the latter of which attach to the plasma membrane to generate forces applied to centrosomes. However, we found that centrosomes were often displaced away from spindle poles in reactive astrocytes. Although this does not necessarily result in abnormal mitosis, an inability of centrosomes to occupy their correct places may have important, initial roles in the development of mitotic arrest. Displaced centrosomes may not be able to orient spindles properly.In mitosis in a normal astrocyte, the migration of centrosomes to spindle poles and the subsequent correct orientation of the spindles strongly depends on interactions between astral microtubules that attach to the subplasmalemmal cytoskeleton of the cell body , 50. HowFurthermore, displaced centrosomes may not be correctly connected to kinetochore microtubules that bind centromeres with centrosomes , 45. ThiThe phenomenon of multiple or fragmented centrosomes exists in a variety of pathological circumstances. It can follow X-irradiation and is a frequent event in malignancies , but neiThe appearance of several spindle pole–like structures during abnormal mitosis indicates that centrosomes, when they are not associated with poles, do not have a leading role in the formation of the mitotic spindle, and that the assembly of kinetochore microtubules is not controlled by centrosomes. In fact, the displacement of centrosomes from spindle poles as well as appearance of several spindle poles may not inevitably leads to arrested mitosis. Abnormal spindles with displaced centrosomes and several pole–like foci of microtubule convergence may be able to perform chromosome segregation after satisfaction of SAC. Thus, an essential role of centrosomes in spindle formation has been reevaluated and may differ in different types of cells .We found that many of the regulatory components of mitotic organization were mis-localized in the abnormal mitoses. These included gamma-tubulin, NUMA, Aurora A and B kinases, BuBR1 and BUB3. In mitosis, Aurora A associates with TPX2, which targets the kinase to spindle microtubules . In the A lack of phosphorylation of Aurora A substrates may also cause mis-localization of critical molecules. For example, one of the substrates of Aurora A kinase is NUMA, and the inactivation of Aurora A causes a loss of NUMA at the cell periphery and an increase at the spindle poles . AnotherThe roles of Bub3 and BubR1, which have been investigated via knockouts and mutations, principally revolve around promoting the attachment of kinetochores to spindles and organizing chromosomes in normal congression . In the One of the characteristic pathologies we observed was a failure of cytokinesis, leading to multinucleated cells. This is a highly complex process, involving the formation of a cleavage furrow and then the ingression of the furrow, followed by abscission and separation of the two daughter cells. One of the proteins required for cytokinesis is Aurora B kinase, which is initially localized to the central spindle and then to the furrow along with Mklp1, a kinesin protein, which Aurora B phosphorylates. In v-Src transformed cells, which display cytokinesis failure, both Aurora B and Mklp1 are mis-localized away from the furrow , 46. MulIn cancer cells, multipolar cell divisions can result in failure of cytokinesis, leading to multinucleated cells . HoweverWe speculate that an additional factor that may underlie mis-localization of critical proteins and chromosomes is the large accumulation of cytoplasmic proteins typical for reactive astrocytes, especially the intermediate filament proteins, GFAP, vimentin, and nestin, and the small heat shock protein, alphaB-crystallin. Our observations suggest that the intermediate filaments are depolymerized during mitosis, which is a normal event. But large amounts of cytosolic proteins could mechanically interfere with spindle microtubules and the movement of chromosomes. Indeed, GFAP immunostaining revealed the protein localized throughout the astrocyte cytoplasm, even surrounding chromosomes and appearing in the spindle, localizations from which it is normally excluded during mitosis.A similar dispersed localization among chromosomes is present for alphaB-crystallin. This small heat shock protein can associate with tubulin and, in vitro, regulate tubulin assembly into microtubules , 24. SpeGFAP, which lead to a massive accumulation of GFAP and small heat shock proteins with the formation of proteinaceous aggregates, Rosenthal fibers. In Alexander disease and its mouse models, many astrocytes display abnormal, C-like mitoses with formation of multinuclear, polyploidal cells and large, multinuclear (2–3 nuclei) astrocytes [High levels of GFAP and small heat shock proteins associated with abnormal chromosome congression and segregation are present in astrocytes of Alexander disease, a severe neurological disorder caused by dominant mutations in trocytes . These suclear 2– nuclei atrocytes , 48.We have used the term “abnormal” mitosis throughout this work, realizing that it does not have a specific meaning, but rather refers to a number of pathological features that include the abnormal positioning of chromosomes in the spindle and a mislocalization of critical mitotic molecules. We have illustrated these abnormalities in several Figures:We consider that the main reason for the arrested mitoses is a failure to form mitotic spindles that are able to perform the normal congression of chromosomes into a metaphase plate. This results in an arrest at the prophase - metaphase transition and the consequent mis-distribution of chromosomes into a large volume. Later on, these chromosomes are enclosed by envelopes and give rise to micronuclei. Several factors contribute to these abnormal mitoses. First, the accumulation of proteins that can interfere with microtubule assembly and the interactions of microtubules with substrates will impede spindle formation. Second, the marked changes in the location and distribution of many proteins participating in mitosis  impedes normal mitosis. Third, the complex cellular geometry in which many enlarged processes are unequally distributed along the cell body and the distribution of centrosomes into proximal processes will produce non-symmetrical pulling-pushing forces produced by astral microtubules that interact with the plasma membrane at various distances from the cell body, interfering with the formation of a normal spindle. See Fig. We found that multinucleated astrocytes could be observed up to a year after an initial excitotoxic insult. While we cannot exclude the possibility that some of the multinucleated astrocytes eventually die, our results support that micronuclei fuse into larger ones, resulting in multinucleated cells with fewer, larger nuclei over time . Nuclear1) Abnormal mitoses are common in proliferating astrocytes reacting to a variety of brain insults, but possible mechanisms underlying this pathology have remained unknown.2) Arrested mitoses appear due to abnormal formation of mitotic spindles.3) Several factors predispose to the formation of abnormal spindles, including changes in cell geometry and the accumulation of large amounts of cytoplasmic proteins.4) Multinucleated astrocytes can survive long periods of time and even re-enter mitosis.Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.


Equipment and hospital surfaces constitute a microbial reservoir that can contaminate hospital users and thus create an infectious risk. The aim of this work, which was carried out for the first time at a hospital in Meknes , is to evaluate the microbiological quality of surfaces and equipment in three potential risk areas .  This study was carried out over a period of 4 months (February–May 2017). A total of 60 samples were taken by swabbing according to the standard (ISO/DIS 14698-1 (2004)) in an environment of dry area and equipment after biocleaning. Isolation and identification were performed according to conventional bacteriological methods and by microscopic observation for fungi. p value <0.001), 13% for the sterilization service, and 7% for the operating room. 89% of the isolates were identified as Gram-positive bacteria against 11% for fungi . Bacterial identification showed coagulase-negative staphylococci (32%), Bacillus spp. (16%), Corynebacterium (8%), and oxidase-negative Gram-positive bacillus (40%) while fungal identification showed Aspergillus niger (n = 2) and Aspergillus nidulans (n = 1).  The study showed that 40% of surface samples were contaminated after biocleaning. The burn unit recorded a percentage of 70% contamination ( To control the infectious risk related to equipment and hospital surfaces, it would be necessary to evaluate the disinfection protocol applied in these units. Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa [Clostridium difficile [Klebsiella pneumoniae [Staphylococcus aureus [Acinetobacter baumannii [Healthcare-associated infections (HAIs) represent a serious public health problem as they lead to increased mortality, morbidity, and costs for patients, their families, and health systems , 2. Accoruginosa , 6. The ruginosa –9. The fruginosa . The hosruginosa  or the eruginosa . Weber eruginosa  reportedruginosa –16. Thusruginosa . Microbiruginosa , while truginosa , 20. Manifficile , Klebsieeumoniae , 22, Stas aureus , 23, coas aureus , and Aciaumannii , 14. So,We carried over a period of 4 months (February–May 2017) the samples from surfaces and medical devices used in three services in a hospital at Meknes (Morocco): burn unit, operating room, and sterilization service. The choice of burn unit was based on the fact that patients hospitalized are immunocompromised and on the result of a study that showed the predominance of clinical bacteria in this service . The cho2 was swabbed and repeated striations in two directions at right angles to each other was carried out in a close zigzag pattern while rotating the swab during sampling of surface ISO 14698-1 (2004) [The microbiological sampling of the surfaces was carried out by the swabbing technique as recommended by the standard ISO 14698-1 (2004) . Samplin1 (2004) . Then, t1 (2004) . The col1 (2004)  recommen2).Using swabs, we have seeded the suspension on a Petri dish containing the PCA (Plate Count Agar) for the enumeration of microorganisms. Then, the Petri dishes were incubated at 37°C for 24 h to 48 hours. After 24 h of incubation, a number of colonies were noted (the number of colonies was presented in colony-forming unit (CFU)/25 cmμL of each sample was inoculated on semiselective media: MacConkey (Enterobacteriaceae), Chapman (staphylococci and micrococci), Cetrimide agar (Pseudomonas aeruginosa), and Sabouraud (fungi). The Petri dishes of bacteria were incubated for 24 hours, as for the fungi for 5 to 7 days. The identification of bacteria was carried out according to conventional bacteriology methods. Fungi (molds) were identified by two fundamental examinations: macroscopic and microscopic [For isolation, a volume of 10 roscopic .p value< 0.001, p value <0.01, and p value <0.05 were considered highly significant, very significant, and significant, respectively.Data entry was done using Microsoft Office Excel 2010. The descriptive and analytical parts were realized using the XLSTAT extension. To give meaning to the results, we used the chi-square test; the n = 24) of the samples analyzed were found to be positive and 60% (n = 36) were found to be negative. The burn unit (BU) department recorded a very high percentage (70%), followed by the sterilization room (S) with a percentage of 13% and finally the operating room with a percentage of 7% .From a total of 60 samples, 40% (ge of 7% . The disn = 2) of the molds were found in the BU, whereas only 33% (n = 1) of the samples showed the presence of the fungal flora in the sterilization room (SR). A percentage of 89% (n = 25) of the isolates were bacteria, while fungi accounted for only 11% (n = 3). The distribution of the positive samples according to the germs is highly significant . Bacterial identification revealed that all bacteria are Gram-positive.The fungal flora is present in two departments, 67% (n = 8) of Gram-positive cocci were identified as coagulase-negative staphylococci, of which 8% were identified as Staphylococcus epidermidis and 4% were Staphylococcus saprophyticus, 16 % of bacteria (n = 4) were identified as Bacillus spp., while Corynebacterium spp. were identified with a percentage of 8% (n = 2), and finally, 40% of Gram-positive bacillus were unidentifiable (n = 10) (They are distributed as follows: 32% ((n = 10) .df = 2, p value <0.01). From the isolates, only 11 strains have a nosocomial interest: 8 coagulase-negative staphylococci and 3 Aspergillus spp. Fungal floras include Aspergillus niger (n = 2) (Aspergillus nidulans (n = 1)   and Aspe (n = 1) .The hospital environment, especially surfaces, is generally colonized by many opportunistic and pathogenic microorganisms. The surfaces are contaminated by microorganisms derived from the patient himself or sedimentation of particles in the air . SeveralNevertheless, studies have shown this relationship , 15. Mic2 > 30) [2 >10) according to the Association for the Prevention and Contamination Study (APCS).The total microorganisms count in the sterilization service showed that the acceptability limits were exceeded (CFU/25 cm2 > 30) . We alsoAspergillus niger (sterilization service and patient's room) and Aspergillus nidulans (patient's room). This result requires a review of hygiene procedures in these services [Aspergillus spp. result in significant mortality and morbidity. Aspergillus niger has been associated with pulmonary disease [In this study, bacteria represent 89% of isolates against 11% for fungi. Several studies have reported contamination of the hospital environment with fungi , 37. Ideservices . Infecti disease , cutaneo disease , and oto disease .Bacillus spp. at 16%, and Corynebacterium spp. at 8%. The medical devices of the Iranian hospitals were colonized by coagulase-negative staphylococci [Bacillus spp. [Bacillus spp. bacteria are constantly present in the environment and do not appear to be accessible to biocleaning, and this could be explained by their ability to sporulate [Our result showed the presence of only Gram-positive bacteria. Other studies have shown the presence of Gram-positive bacteria and Gram-negative bacteria , 37, 41.ylococci . The resylococci . Surveilylococci  and Bacilus spp. , 37. Meuporulate . The preporulate  includinporulate . Microorporulate , and . The identification revealed the exclusive presence of Gram-positive bacteria with a preponderance of coagulase-negative staphylococci and the presence of fungal agents of


Gut microbiota may contribute to the pathogenesis and development of autism spectrum disorder. The maternal gut microbiota influences offspring gut microbial structure and composition. Lachnospiraceae ASVs from CAG15 with their mothers was significantly lower than that of children with TD. Moreover, we found that CAG12, CAG13, and CAG18 negatively correlated with the severity of social deficits in ASD children. ASD children who shared any one of the four  ASVs in CAG13 and CAG18 with their mothers showed a lower level of social deficits than ASD children that did not share those with their mothers. These data demonstrate that these shared gut bacteria in ASD children are associated with their developmental level and social deficits. This work provides a new direction toward understanding the role of the gut microbiota in the pathogenesis and development of ASD. The gut microbiota of autism spectrum disorder (ASD) children differs from that of children without ASD. The maternal gut microbiota impacts offspring gut microbiota. However, the relationship between the development of ASD and gut bacteria shared between children and their mothers remains elusive. Our study recruited 76 children with ASD and 47 age- and gender-matched children with typical development (TD), as well as the mothers of both groups, and investigated their gut microbiota using amplicon sequence variants (ASVs). The gut microbiota of ASD children was altered compared with that of children with TD, while no significant alterations were found in their mothers. We established 30 gut bacterial coabundance groups (CAGs) and found the relative abundances of CAG15 and CAG16 significantly decreased in ASD children. CAG15 showed a positive correlation with developmental level. The proportion of ASD children who shared either one of the two IMPORTANCE Gut microbiota may contribute to the pathogenesis and development of autism spectrum disorder. The maternal gut microbiota influences offspring gut microbial structure and composition. However, the relationship between the clinical symptoms of autism spectrum disorder and the gut bacteria shared between children and their mothers is not yet known. In our study, the gut microbiota of children with autism spectrum disorder differed from that of children with typical development, but there were no differences in the gut microbiota of their mothers. More importantly, gut bacteria shared between children with autism spectrum disorder and their mothers were related to developmental disabilities and social deficits. Thus, our study suggests that these shared gut bacteria may play an important role in the development of autism spectrum disorder. This provides a new direction for future studies aiming to explore the role of the gut microbiota in autism spectrum disorder. Autism spectrum disorder (ASD), first described by Leo Kanner in 1943, is a set of neurodevelopmental disorders . Develop4–Bacteroides fragilis NCTC 9343 was reported to reduce the level of chronic inflammation in the offspring of mice with immune activation, which alleviated ASD-like behavioral abnormalities such as defects in communication and stereotypic behaviors (Lactobacillus reuteri MM4-1A (ATCC PTA-6475) ameliorated the ASD-like behavioral abnormalities in offspring of obese mice via oxytocin stimulation  were recruited to the study along with 47 age- and gender-matched children with typical development (TD) and their mothers (MT). The gut microbiota of these groups were analyzed by sequencing the V3-V4 regions of the bacterial 16S rRNA gene in fecal samples. Gut microbiota of children with ASD differed from that of children with TD, but no significant differences were found between the gut microbiota of their mothers. Gut bacteria shared between children with ASD and their mothers were found to be associated with ASD behavioral symptoms.1, vitamin C, Na, Ca, Mg, Fe, and Zn were found between the ASD and TD groups  were screened using the childhood autism rating scale (CARS) and were diagnosed with ASD based on the autism diagnostic interview (ADI-R) and autism diagnostic observation schedule (ADOS) see , and theoups see . Howeveroups see . The ASDTD group . The GI TD group . The proTD group . DevelopTD group , suggest10.1128/mSphere.01044-20.1TABLE S1n = 76; ADI-R, autism diagnostic interview, n = 76; communication, n = 76; VC, verbal communication, n = 47; NVC, nonverbal communication, n = 29; RSI, reciprocal social interaction, n = 76; RBSP, repetitive behavior and stereotyped patterns, n = 76; ADOS, autism diagnostic observation schedule, n = 76; ASD, autism spectrum disorder. The data for CARS, ADI-R, and ADOS were measured as in Li et al.’s study . CARS, childhood autism rating scale, Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.01044-20.2TABLE S2t tests were performed to analyze the variations between the ASD and TD groups. **, P < 0.01; TD, typical development; ASD, autism spectrum disorder. Download Table S2, DOCX file, 0.02 MB.Dietary intake of the ASD and TD groups for 24 h before the collection of fecal samples. Data are shown as means ± SEMs. Student’s Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.01044-20.3TABLE S3n of children in the ASD and TD groups. Pearson chi-square tests were used to analyze differences. **, P < 0.01; ***, P < 0.001; ASD, autism spectrum disorder; TD, typical development. Download Table S3, DOCX file, 0.02 MB.GI symptoms in the ASD and TD groups. Data are presented as Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.01044-20.5FIG S1P < 0.001 by Student’s t test; TD, typical development, n = 42; ASD, autism spectrum disorder, n = 70. Download FIG S1, DOCX file, 0.2 MB.A higher level of GI severity in ASD children. The boxes represent the interquartile ranges, lines inside the boxes stand for medians, and whiskers represent the minimum and maximum values. ***, Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Gut microbial alterations in ASD children and their mothers (MA) (28 to 41 years old) were explored in comparison with those in children with TD and their mothers (MT) (30 to 40 years old) by sequencing the bacterial 16S rRNA gene V3-V4 regions in fecal samples from these groups. Totally, 5,755,973 high-quality sequencing reads  were denoised into 3,642 amplicon sequence variants (ASVs). Richness (observed species) of the gut microbiota in the ASD group was significantly greater than that in the TD group . There wP = 0.002 with permutational multivariate analysis of variance [PERMANOVA], 9,999 permutations), but there was no significant difference in the gut microbial structures between the MA and MT groups . Based on Bray-Curtis distance, we showed that the similarity of gut microbial structures between ASD children and their mothers was higher than that between children with TD and their mothers  of the Bray-Curtis distance based on the ASV data revealed that the overall structure of the gut microbiota in the ASD group significantly differed from that in the TD group   and MT versus MA (B) based on Bray-Curtis distance. Data were processed by log(10) transformation. TD, children with typical development, Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.01044-20.7FIG S3P < 0.01 by Student’s t test; TD, typical development, n = 47. ASD, autism spectrum disorder, n = 76. Download FIG S3, DOCX file, 0.3 MB.Higher similarity of gut microbial structures between ASD children and their mothers than between TD children and their mothers. Bray-Curtis distance was used to calculate the distance between values for children and for their mothers. The boxes represent the interquartile ranges, lines inside the boxes stand for medians, and whiskers represent the minimum and maximum values. **, Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Lachnospiraceae, while CAG16 predominantly comprised ASVs from the family Lachnospiraceae and the genus Bacteroides  in 30 coabundance groups (CAGs). Download Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.01044-20.8FIG S4n = 47; ASD, autism spectrum disorder; n = 76. Download FIG S4, DOCX file, 1.2 MB.Relative abundances of CAGs between the ASD and TD groups. The boxes represent the interquartile ranges, lines inside the boxes stand for medians, and whiskers represent the minimum and maximum values. Mann-Whitney test was used to analyze the variations between the two groups. CAG, coabundance group; TD, typical development, Copyright © 2020 Chen et al.2020Chen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The relationship of gut microbiota to GI severity and developmental level, which were significantly altered in the ASD group, was examined by calculating Spearman’s correlation coefficients in gut bacterial CAGs versus the GI severity index and CAGs versus GDS in the ASD and TD groups. None of the CAGs showed a significant correlation with the GI severity index, while the relative abundance of CAG15 showed a significant positive correlation with DQ scores of adaptive behavior, gross motor, fine motor, language, and personal social behavior, the five parameters of the GDS .Lachnospiraceae ASV3491 and Lachnospiraceae ASV790  in the ADI-R . This sulinsella .Ruminococcaceae ASV1617, Lachnospiraceae ASV2314, and Ruminococcaceae ASV330 were all located in CAG13  group, while ASD children who did not share the ASV with their mothers were annotated as “no-shared” the ASV (NS plus ID of the ASV) group. For example, ASD children who shared ASV1617 with their mothers were annotated as the S1617 group, and ASD children who did not share the ASV with their mothers were annotated as the NS1617 group. The social deficits of these S groups and their NS groups were then compared. The S1617, S2314, and S330 groups had significantly decreased scores of communication compared with those for the NS1617, NS2314, and NS330 groups, respectively , and C. in CAG13 . In addiin CAG13 , and ColIn summary, we found specific gut bacteria that were shared between ASD children and their mothers and which related to social deficits.In this study, we found an altered gut microbiota in ASD children compared with that in age- and gender-matched children with TD, while there were no significant differences with the gut microbiota of their mothers. The similarity of gut microbial structure between ASD children and their mothers was higher than that between children with TD and their mothers. Furthermore, specific gut bacteria shared between children and their mothers were associated with neurodevelopmental level and social deficits in children with ASD.Lachnospiraceae and the genus Bacteroides, decreased in ASD children. These results were consistent with previous studies that reported the abundances of this family and genus were decreased in Chinese ASD children  of histone H3 at lysine 23 in mouse embryos and fibroblasts and human embryonic kidney 293 cells, and this deficiency was also found in children with neurodevelopmental delay (Ruminococcaceae produce acetate (–Bifidobacterium contains gamma-aminobutyric acid (GABA)-producing bacteria . This clinical trial recruited 184 children and mothers of 123 of these children. Participants in our study comprised 123 children and their mothers, while participants in Li et al.’s study were the 184 children .Seventy-six children with ASD and their mothers were recruited to the trial. The children were screened by CARS and diagnosed with ASD by ADI-R and ADOS in the Children’s Mental Health Research Center of the Nanjing Brain Hospital affiliated with Nanjing Medical University. Then, 47 children with TD from Ying Hua kindergarten (Nanjing) and their mothers were recruited. Children with ASD who suffered from other neurological conditions, such as epilepsy and attention deficit hyperactivity disorder (ADHD), and serious organic diseases, such as hepatorenal function disorder, were excluded. Children with TD were confirmed by at least two clinicians as not having any neurodevelopmental disorders, serious organic disease, such as hepatorenal function disorders, or any gastrointestinal surgery.Clinical parameters, including general anthropometric parameters, GDS, CARS, ADI-R, and ADOS, of children were measured based on the methods described in previous studies , 48, 49.Tools for fecal collection were provided to participants who had not suffered from fever or used antibiotics or any other medications for more than 3 days in the previous month. Maternal fecal samples were collected the next day, and samples from children were collected within 1 week of the psychological assessments. Fecal samples stored in stool collection tubes with DNA stabilizer  were collected in participants’ homes by their mothers (children) or themselves. The tubes with DNA stabilizer protect gut bacteria in fecal samples at room temperature for at least 24 h. Fecal samples received at the Children’s Mental Health Research Center of the Affiliated Brain Hospital of Nanjing Medical University were immediately stored at −80°C until fecal DNA extraction.Gut microbial DNA in fecal samples was extracted using methods reported in previous studies. An Illumina MiSeq platform  with MiSeq reagent kit v3 (600-cycle)  was used to sequence the 16S rRNA gene V3-V4 regions of gut bacteria in fecal samples. A two-step amplification (amplification of 16S rRNA gene V3-V4 region and the index PCR) was used to prepare the library according to the manufacturer’s instructions  with modifications described by Zhang et al. .Sequence data from the Illumina MiSeq platform were inputted to QIIME2 as type “SampleData[PairedEndSequencesWithQuality],” and all steps of sequence processing and quality control were performed in QIIME2 (version 2018.06). Adapter sequences were trimmed by using the script “qiime cutadapt trim-paired.” The DADA2 pipeline, including error filtering, trimming, denoising, merging of paired reads, and removal of chimeras, was used to cluster high-quality reads into amplicon sequence variants (ASVs) which were annotated using the SILVA reference database (version 132). These reads of each sample were downsized to 10,000 to normalize the even sampling depths.R) based on their relative abundance. This R matrix, also known as topological overlap matrix (TOM), was converted to a correlation distance (1 − R) matrix, also known as dissTOM, in the R (version 3.5.1) package WGCNA , were used to analyze the clinical parameters of volunteers. Permutational multivariate analysis of variance (PERMANOVA) was utilized to evaluate the significance of the differences in gut microbial structures in ASD, TD, MA, and MT groups in QIIME2. To adjust the P value for multiple testing, false-discovery rate (FDR) values were estimated using the Benjamini-Yekutieli method in correlation analysis between CAGs and clinical parameters with Spearman’s algorithm. P values of <0.05 were regarded as statistically significantly different.Student’s PRJNA644763.The 16S rRNA V3-V4 amplicon sequencing data are available in the NCBI Short Read Archive (SRA) repository under accession number


The biosafety concern of silica nanoparticles (SiNPs) is rapidly expanding alongside with its mass production and extensive applications. The cardiovascular effects of SiNPs exposure have been gradually confirmed, however, the interaction between SiNPs exposure and atherosclerosis, and the underlying mechanisms still remain unknown. Thereby, this study aimed to explore the effects of SiNPs on the progression of atherosclerosis, and to investigate related mechanisms.−/− mice fed a Western diet. Ultrasound microscopy showed a significant increase of pulse wave velocity (PWV) compared to the control group, and the histopathological investigation reflected a greater plaque burden in the aortic root of SiNPs-exposed ApoE−/− mice. Compared to the control group, the serum levels of total triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) were elevated after SiNPs exposure. Moreover, intensified macrophage infiltration and endoplasmic reticulum (ER) stress was occurred in plaques after SiNPs exposure, as evidenced by the upregulated CD68 and CHOP expressions. Further in vitro, SiNPs was confirmed to activate ER stress and induce lipid accumulation in mouse macrophage, RAW264.7. Mechanistic analyses showed that 4-PBA (a classic ER stress inhibitor) pretreatment greatly alleviated SiNPs-induced macrophage lipid accumulation, and reversed the elevated CD36 expression induced by SiNPs.We firstly investigated the in vivo effects of SiNPs exposure on atherosclerosis via intratracheal instillation of ApoE−/− mice, which was related to lipid accumulation caused by ER stress-mediated upregulation of CD36 expression in macrophage.Our results firstly revealed the acceleratory effect of SiNPs on the progression of atherosclerosis in ApoE The rapid development and enormous progress in nanotechnology bring the toxicological concerns to nanomaterials (NMs), which might pose potential threats to human health and the environment. Silica nanoparticles (SiNPs) rank in the top two global productions in NMs, with an annual output of nearly 1.5 million tons , 3. Such2) NPs [Despite the most important route of exposure to SiNPs being inhalation, especially in occupational settings, cardiovascular system is a principal site of extra-pulmonary toxic effects of SiNPs . It has 2) NPs . Similar2) NPs . It was 2) NPs , and car2) NPs . However2) NPs , probabl2, nickel hydroxide and indium oxide NPs could accelerate the pathological progress of atherosclerosis [−/−) mice fed a Western diet and conduct in vitro experiments for mechanism research. The current study may provide persuasive evidence for safety evaluation and risk management of SiNPs, and offer a new insight into the mechanisms underlying the adverse effects of SiNPs on cardiovascular system.Atherosclerosis is the major cause of cardiovascular diseases (CVDs), and its global burden is projected to rise substantially in the next few decades, particularly in developing low- and middle-income countries , 20. Exiclerosis –24. But clerosis , and alsclerosis . So far,clerosis . In contclerosis . Owing tSiNPs were spherical in shape and uniform in size as manifested by the scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images Fig. a, b. The−/− mice fed a Western diet. Moreover, the ultrasound biomicroscopy (UBM), a useful tool for the non-invasive, dynamic characterization of blood vessels in animal models, was applied to monitor the vascular morphology and elasticity, and repeated measurement data was acquired. The experimental design was shown in the Fig. −/− mice.The effect of SiNPs on atherosclerosis was firstly evaluated by using in vivo model, which was achieved by intratracheal instillation of SiNPs on ApoEp < 0.05). Oil-Red O staining revealed a large amount of lipid deposition inside the plaque and a slight increase trend of lipid content after SiNPs exposure but no significance in comparison to the control group, probably attributing to the higher plaque area in SiNPs-treated mice , in despite of no significant difference in total cholesterol (TC) and high density lipoprotein cholesterol (HDL-C) levels , low-density lipoprotein cholesterol (LDL-C) levels and calculated atherogenic index (AI), and declined HDL-C/LDL-C ratio, especially at the dose of 6.0 mg/kg·bw group (The macrophage infiltration and endoplasmic reticulum (ER) stress inside the lesion were assessed by immunohistochemistry. As shown in Fig. −/− mice model, a murine macrophages cell line, RAW264.7 was used for the further in vitro investigations. As depicted in Fig. Macrophage is a major cell type involved in the onset and progression of atherosclerotic plaque, especially plays a crucial role in the regulation of inflammatory response and foam cell formation. In consideration of the observed promoting effect of SiNPs on the progression of atherosclerotic plaque in ApoEMacrophage-derived foam cell formation is a hallmark event during the progression of atherosclerotic lesion. Oxidized LDL (oxLDL) was applied to construct a foam cell model in vitro so as to explore the role of SiNPs in the formation of macrophage-derived foam cells. As shown in Fig. The intracellular lipid homeostasis in macrophage was precisely regulated by lipid influx and efflux. Thus, we further detected the expressions of factors controlling the cholesterol uptake (CD36 and SRA1) and its efflux  in RAW264.7 cells. Real-time PCR results showed that SiNPs exposure caused the up-regulated mRNA expressions of CD36 and SRA1, while down-regulated mRNA expressions in ABCA1, ABCG1, and SRB1. In addition, SiNPs had no influence on the mRNA level of ACAT1, a critical regulator to re-esterify excessive free cholesterol to cholesterol ester to store in lipid droplets . Further−/− mice with SiNPs through intratracheal instillation for 12 weeks by mimicking occupational scenario, and UBM was applied to monitor the development and progression of atherosclerosis. Currently, only few studies have reported the vascular effects of NPs through Doppler ultrasound trace [With the mass production and application of SiNPs, its biological safety issues, especially on the health effects of nano-handling workers need to be considered. Atherosclerotic related CVDs is the leading cause of mortality worldwide. So far, there is still no conclusive information on the pro-atherogenic potential of occupational SiNPs exposure. To the best of our knowledge, this is the first in vivo study to confirm SiNPs exposure could affect the progression of atherosclerosis. We firstly administered Western diet-fed ApoEnd trace . As a rend trace . PWV hasnd trace . Besidesnd trace , which wnd trace .−/− mice as indicated by the histological analysis of aortic root. According to the American Heart Association’s definition of human atherosclerotic stages [On the basis of a clearly observed plaque and an increased PWV value in SiNPs-treated mice, the experiment was terminated and atherosclerotic burden was assessed by histopathological staining. In despite of no difference on the plaque distribution in the whole aorta, SiNPs exposure triggered a greater plaque burden in ApoEc stages , 37, thec stages , 38–40 ac stages , and foac stages . Moreovec stages . In addic stages . However2 NPs in ICR or ApoE−/− mice, ultimately contributing to the initiation and progression of atherosclerosis [2 NPs affected lipid metabolism, as evidenced by a higher level of serum LDL when compared to the normal physiologic range for LDL in adults in China [−/− mice, characterized with the increased serum levels of TG and LDL-C. To be noted, the increase in serum LDL-C content was positively correlated with the plaque area in aortic root. Meanwhile, the AI index was increased, whilst HDL-C/LDL-C ratio was decreased in SiNPs group when compared with the control group, hinting the dyslipidemia caused by SiNPs may contribute to the progress of atherosclerotic lesions [−/− mice, but no alteration of lipid profile [Lipid is one of the most important stimuli initiating atherogenesis and the plasma lipoproteins are involved in foam cell formation and inflammatory regulation within plaques –45. The clerosis , 47, 48.in China . In agre lesions , 50. Coi lesions . Besides lesions . Intrigu profile , 24. It  profile .ER stress, also known as unfold protein response (UPR), plays a crucial role in the pathogenesis of a series of cardiovascular disorders, including atherosclerosis, ischemia . ER is aMacrophage-derived foam cell is the crucial determinant of the initiation and progression of atherosclerosis lesion , contrib−/− mice. And also, it is novel in the utilization of ultrasonic technique to non-invasive, dynamic assess the vascular function and plaque formation. Consequently, the current study demonstrated an increased plaque size, macrophage infiltration and ER stress in the aortic root after long-term SiNPs exposure via intratracheal instillation, accompanied by the aggravated hyperlipidemia and artery stiffness in plaque. The phenomena mentioned above was significantly manifested in the SiNPs group with a higher dose (6.0 mg/kg·bw), which was closely associated with the exposure mode . Specific details about the dose selection and conversion can be seen in Method part. Moreover, a comprehensive molecular mechanism related to the promotion of atherosclerotic progression by SiNPs was provided. That was, ER stress-mediated up-regulated CD36 expression was validated to be a major contributor of SiNPs to promote foam cell formation and ultimate plaque progression. Overall, our results provide new insight into the cardiovascular toxicological effects of SiNPs, and inhibition of ER stress might be a promising approach to alleviate NPs-induced vascular lesion. Besides SiNPs, other NMs, e.g. carbon nanotubes, TiO2 NPs, ZnO NPs, were found to have pro-atherogenic potential, ultimately leading to the onset and development of CVDs. Hence, NPs exposure may aggravate the cardiovascular risk of occupational population, and more efforts should be made for ensuring occupational safety, and also for a reasonable and safer application of nanoproducts.In summary, this is the first study to directly address the acceleratory effect of SiNPs in atherosclerotic plaque progression by using ApoEThe amorphous SiNPs used in the experiments were prepared by the Stöber method as previously described . The par−/− mice at the age of 1–2 months is commonly used as the animal model for spontaneous atherosclerosis [−/− mice  were obtained from the Experimental Animal Center of Capital Medical University  to assess the long-term effect of SiNPs in the development of atherosclerosis. All mice were housed in sterilized filter-topped cages with free access to food and water, and maintained in a specific pathogen-free facility with a constant humidity (50 ± 5%), temperature (24 ± 1 °C) at 12/12-h light/dark cycle. After 1 week of acclimation, all the mice were supplied with a Western diet  for a rapid establishment of the murine atherosclerosis model. According to the development stage of atherosclerosis in ApoE−/− mice fed with high-fat diet [ApoEclerosis . Male Apfat diet , the SiN3 [2), 6 mg/m3 was used. Therefore, a worker (60 kg) exposed to a concentration of 6 mg/m3 SiNPs for 8 h (1 workday) without proper protection would result in an approximate pulmonary dose of 0.44 mg/kg·bw  [4 to 2 × 105/cm3, with mass concentrations of > 50 μg/m3 near major highways [3 near major highways  [The applied dose of SiNPs was in reference to an inhalation study in mice , in whic3 . In consn human) , 87. Accn human) , the doshighways . Here, thighways , the inhMice in SiNPs groups were administered SiNPs suspension through intratracheally instillation, once in every 7 days and 12 times in total, whereas the control mice were instilled with 0.9% saline instead. The volume of intratracheal instillation was controlled to be 50 ± 5 μl. Furthermore, the UBM of three mice per group was performed during the experiment. In addition, the body weight and food intake of mice were monitored and weighed weekly  equipped with MS 400/550D mechanical transducers were used, and the ultrasound imaging parameters of LCCA were measured. During the experiment, mice were anaesthetized with isoflurane gas resulting in a heart rate of approximately 500 beats/min, and the hair from the anterior chest wall was carefully shaved, and warm ultrasound transmission gel was liberally applied to ensure optimal image quality. The IMT and PWV were obtained using VEVO LAB software. The EKV two-dimensional dynamic image was analyzed by VEVO VASC software, and indicators representing vascular compliance  were obtained. On the basis of a previous description , IMT wasBlood samples were collected and centrifuged at 3000 rpm, 4 °C for 10 min. The contents of TC, TG, HDL-C, and LDL-C in mice serum were measured by an automatic biochemical analyzer (HITACHI 7180) combining commercial kits . The ratio of HDL-C/LDL-C was calculated, as well as AI according to the formula: AI = (TC - HDL-C)/HDL-C .For lesions throughout the aorta, the whole aortas of three mice per group were separated, cut longitudinally after removing excess adipose tissue, and stained with Oil-Red O staining  for 10 min. Afterwards, the stained aortas were placed in 75% alcohol until the artery wall without lesions was cleaned. Images were captured and analyzed by Image J software. Furthermore, for the lesion at the aortic root, the entire aortic root was immersed in 4% paraformaldehyde for 24 h, embedded into paraffin or optimal cutting temperature (OCT) for histological examination. The cross-sections of the aortic root were stained with H&E, Oil-Red O, Masson and Alizarin Red for the quantification of plaque area, lipid and collagen content, and aortic calcification. It is worth noting that the regional error in lesion size was avoided by acquiring of the sequential cross-sections throughout the entire aortic root as previously described . UltimatImmunohistochemistry was performed in the paraffin-embedded artery root to determine the expressions of CD68 (a macrophage marker), CD36 , Bip and CHOP (biomarkers for ER stress) in situ. Briefly, the dehydrated paraffin sections were immersed in 1 mM EDTA (pH = 9) for antigen retrieval, and incubated with 3% hydrogen peroxide to abolish endogenous peroxidase. The sections were incubated with the primary antibody for CD68 , CD36 , Bip , or CHOP  overnight at 4 °C, and then incubated with the corresponding secondary antibody and stained with 3,3′-diaminobenzidine (DAB). These primary antibodies were diluted with 5% BSA solution at a ratio of 1: 200. Moreover, the nucleus was stained with hematoxylin. Finally, the percentage of positive-staining area in the whole plaque of aortic root was analyzed using the Image J software. All analyses were performed by one investigator blinded for the treatment.The ultrastructure of lesion was observed by using TEM . In brief, the first branch of aorta was fixed by 2.5% glutaraldehyde overnight, rinsed with 0.1 M phosphate buffer, and postfixed with osmic acid for 2 h. After being dehydrated in ethanol with concentration gradients and acetone, the sample was embedded in epoxy resin. Ultimately, the ultrathin sections (50 nm) were obtained and imaged under TEM.2 incubator. SiNPs were diluted by DMEM to appropriate concentrations, and an ER stress inhibitor, 4-phenylbutyric acid  was applied . The dosage of SiNPs was set according to the cell viability analysis by using MTT assay. Since a significant acute toxicity (24 h) was seen in SiNPs-treated group at a concentration of 50 μg/ml, while simultaneously with cell viability > 70%, the exposure mode of SiNPs  was used in the subsequent in vitro experiments. Similarly, the application of 4-PBA was set up through MTT assay and also verified as evidenced by an efficient inhibition on the up-regulated expressions of Bip and CHOP induced by SiNPs. See details in the supplementary Fig. Mouse macrophage cell line, RAW264.7 cells were cultured in DMEM  with 10% fetal bovine serum  at 37 °C in a 5% COAfter 50 μg/ml SiNPs treatment for 24 h, the cellular morphology and alterations of cellular ultrastructure were observed by SEM , and TEM . Based on the TEM image, the particle uptake and internalization were verified by energy dispersive spectrometry .Intracellular lipid droplets were determined by Oil-Red O staining as previously described . In brieThe total cellular RNA was extracted by using a RNAsimple Total RNA kit , and reversely transcribed to cDNA using a PrimeScript™ RT reagent kit . The quantitative PCR was performed by using the SYBR Premix Ex TaqTM II  in a real-time PCR machine . The relative expression in mRNA levels of lipid transport  and esterification (ACAT1), and also ER stress indicators (Bip and CHOP) were quantified. Each experiment was conducted in triplicate with β-actin as the internal standard. Primers used for quantitative PCR analysis were listed in the supplementary file Table .The whole cellular protein was extracted by a Protein Rapid Extraction kit , and quantified by BCA protein assay . After denaturation, protein lysate was separated with SDS-PAGE, and transferred to a nitrocellulose membrane . The membrane was blocked with Tris-buffered saline (TBS) solution containing 5% skim milk powder for 1 h at room temperature. After wash three times with TBST (TBS with 0.05% Tween-20), membrane was incubated with the primary antibody diluted by TBST solution (1: 1000), including Bip , CHOP , CD36 , GAPDH  and β-actin  overnight. After three-time wash with TBST, membrane was incubated with the corresponding fluorescent secondary antibody  for 1 h at room temperature, and ultimately detected using Odyssey® CLx imaging system . At least three independent experiments were performed. The relative expression level of protein was analyzed by Image Studio™ quantification software  with β-actin or GAPDH as internal control, and normalized to the control group.p value < 0.05 indicates statistical significance.Data were expressed as mean ± standard deviation (SD). Significant differences in ultrasound data were analyzed by ANOVA of repeated measurement data. The t-test of independent samples was used to analyze the significant difference of the intracellular cholesterol content and lipid influx/efflux factor expression in RAW264.7 cells, as well as CD36 expression in mouse plaque lesions. Significant differences in the remaining data were analyzed by one-way ANOVA. The LSD test was selected for post hoc test of homogeneous data, whereas Dunnett’s T3 test for the post hoc test of heterogeneous data. A two-tailed Pearson correlation test was applied to determine the correlation between the lesion areas in aortic root and serum lipid levels. All data were analyzed by SPSS 20.0 software, and Additional file 1 Fig. S1. Histopathology alterations of lung tissues after intratracheal instillation exposure to SiNPs in ApoE−/− mice. n = 4 per group. Fig. S2. The body weight and food intake trends of mice. The body weight and food intake of the mice was measured weekly during the experiment. Data are expressed as means ± SD, n = 9. Fig. S3. Method for analyzing plaque area in aortic root. Regional errors in lesion size were avoided by acquiring sequential throughout of the entire aortic root. The lesion with the largest area was used for statistical analysis. Fig. S4 4-PBA inhibited the ER stress induced by SiNPs in RAW267.4 cells. (A) Cell viability was determined by MTT assay after 24 h-treatment with SiNPs. (B) Cell viability was determined after 4-PBA pretreatment .  4-PBA inhibited up-regulation of Bip and CHOP in RAW264.7 cells induced by SiNPs. *p < 0.05 vs Control, #p < 0.05 vs SiNPs treatment group. Data are expressed as means ±SD of three independent experiments. Table S1. Real-time PCR Primer Pairs


The aim of the current study was to evaluate oncologic outcomes of patients who were treated with salvage hysterectomy (HT), compared to systemic chemotherapy (CT) for persistent cervical cancer after definitive radiotherapy (RT)/ concurrent chemoradiotherapy (CCRT).n = 317). Those who underwent a HT for persistent cervical cancer after definitive RT/CCRT were matched with propensity scores for patients who underwent systemic CT. Oncologic outcomes between the two groups using a propensity score matched–cohort analysis were compared.Patients with persistent cervical cancer treated with definitive RT/CCRT at 35 institutions from 2005 to 2014 were reviewed retrospectively , Increasing residual tumor size was significantly associated with a high incomplete resection rate . Severe late adverse events occurred in 7 patients (9.9%) in the HT cohort.A total of 142 patients with persistent cervical cancer after definitive RT/CCRT were included after matching . All background factors between HT and CT groups were well balanced. Median overall survival was 3.8 and 1.5 years in the HT and CT groups, respectively (The current study demonstrated that, when compared to systemic CT, the adoption of salvage HT for patients with persistent cervical cancer after definitive RT/CCRT reduced mortality rate by about 60%. This indicates that salvage HT could be curative treatment for those patients. Further prospective clinical trials with regard to salvage HT after RT/CCRT are warranted.The online version contains supplementary material available at 10.1186/s12885-020-07672-w. The survival of patients with cervical cancer has improved over recent decades due to improvements in screening, which allows for the detection and removal of precancerous lesions, generalization of vaccination against human papilloma virus (HPV), and the improved efficacy of anticancer therapeutics. However, there were approximately 12,820 new cases and 4210 cervical cancer-associated deaths in the US in 2017 [Early stage cervical cancer can be cured with surgery, while concurrent chemoradiotherapy (CCRT) is accepted as the standard treatment for patients with locally advanced cervical cancer . HoweverSalvage hysterectomy (HT) is considered curative treatment; however, very little data exists showing the survival advantage of HT over standard systemic CT. Many guidelines do not have definitive recommendations regarding the treatment of residual tumors of the uterine cervix after radiotherapy (RT)/CCRT, although pelvic exenteration or radical HT is raised as an optional treatment for patients with local recurrence of central disease –8.p = 0.0001) [Ota, et al. showed that HT significantly improved disease-specific 5-year survival rates for patients with residual tumor after RT  . However 0.0001) –15.The aim of the current study is to evaluate oncologic outcomes of patients with persistent cervical cancer after definitive RT/CCRT who were treated with salvage HT, compared to systemic CT using a propensity score analysis.We conducted a retrospective cohort study using data from 35 academic and/or regional cancer centers that belong to the Japan Clinical Oncology Group (JCOG), the largest Japanese cooperative group funded primarily by the National Cancer Center Research and Development Fund. The protocol was approved by the institutional review board of each participating institution, and the need for written informed consent from each patient was waived.Patients were identified by using an institutional database at each participating institution. All patients were treated with definitive RT/CCRT between January 2005 and December 2014. To be included in this study, patients had to meet all of the following conditions: (1) persistent cervical cancer clinically diagnosed after definitive RT/CCRT; (2) salvage treatment with HT, excluding pelvic exenteration (PE), or systemic CT within a year after completion of definitive RT/CCRT; (3) clinical stage IB1, IB2, IIA1, IIA2, IIIA, IIIB and IVA at initial treatment, according to the staging system of the International Federation of Gynecology and Obstetrics (FIGO); (4) less than 75 years of age at the diagnosis of persistent cervical cancer; (5) histological confirmation of the primary site; (6) histological type of squamous cell carcinoma (SCC), adenocarcinoma (AC), or adenosquamous cell carcinoma (ASC).For eligible patients, we noted data regarding the following clinical and pathological details: patient demographics, treatment methods of definitive RT/CCRT, status of persistent cervical cancer, treatment contents and adverse events of salvage HT or systemic CT, and survival outcomes. For patient demographics, patient age and performance status (PS) at diagnosis of persistent cervical cancer, histological type, FIGO stage, tumor diameter, presence or absence of invasion to corpus lesion and number of lymph node metastasis at initial treatment were collected. Treatment methods of definitive RT/CCRT were evaluated based on total RT dose to the cervical lesion, presence or absence of intracavitary brachytherapy (ICBT) and concurrent chemotherapy. All treatments were normalized to biologically equivalent total doses in 2 Gy per fraction (EQD2) using the linear-quadratic model with α/β = 10 Gy for the tumor. Total RT doses were calculated by summation of each EQD2, external beam radiotherapy (EBRT), excluding the fractions with central shielding and ICBT, prescribed at point A. Regarding the status of persistent cervical cancer, we recorded tumor diameter, presence or absence of invasion into the parametrium or vaginal wall, the number of residual lymph node metastases and the period between completion of definitive RT/CCRT and diagnosis of persistent cervical cancer. Salvage HT was abstracted for mode of hysterectomy , operation time, estimated blood loss and status of residual tumor at the end of surgery. Systemic CT was abstracted for the drugs to be used and response to the treatment assessed according to RECIST version 1.1. For survival outcomes, progression-free survival (PFS) and OS were recorded.Cancer stage was re-classified based on the 2008 FIGO staging . DefinitP < 0.05 was considered statistically significant . The cut-off value regarding complete resection of persistent cervical cancer in a HT group was evaluated using receiver operating characteristic (ROC) analysis. Statistical package for Social Science software  and R  were used for all the analyses.We defined two treatment groups: a HT group and a CT group. The former comprised patients who underwent salvage HT for persistent cervical cancer and the latter included patients who received systemic CT. To control for confounders, the propensity score for selecting treatment was estimated by a multivariate logistic regression model, with treatment groups as dependent variables, and all the variables listed in Table Between 2005 and 2014, we identified 309 patients from 35 institutions who underwent salvage HT or systemic CT; 298 patients were included in the analysis . Overall mortality was reduced in the HT cohort, with an estimated HR of 0.41 . PFS for patients in the HT cohort was also significantly longer than that of patients in a CT cohort . Overall risk of recurrence was reduced in the HT cohort, with an estimated HR of 0.45 .The median follow-up period for OS was 4.6 years in the propensity score matched data set. Figure A flow chart in supplementary Figure AP < 0.001)   Fig. a. The HR01) Fig. b. HR of P = 0.500, HR 0.653, 0.198–2.158)   Fig. a. MedianP = 0.016, OR1.11, 95% CI 1.02–1.22). With reference to pathological R0, both uni and multivariate analysis revealed that increasing tumor size of persistent cervical cancer alone was significantly associated with a high incomplete pathological resection rate . The cut-off values of the persistent tumor diameter for R0 resection and pathological R0 resection after ROC analysis were 40 mm  and 11 mm , respectively . Based on the results of that study, the Japanese Clinical Oncology Group is now planning to conduct a multicenter prospective trial to address this issue.Additional file 1 : Figure A1. Propensity score distributions (A) before matching, and (B) after matching. HT, hysterectomy; CT, chemotherapy. Figure A1A demonstrates an initial dissimilarity across the two cohorts on the basis of the propensity score distributions. However, after matching, the distributions of the propensity score for the two cohorts  and 11 mm for pathological R0 resection (B). Figure A4. Survival comparison using propensity matched data between salvage HT and systemic CT for patients with persistent cervical cancer after RT/CCRT. Patients with stage IB1 and IVA malignancies were excluded from the analysis. Figure A5. Survival comparison using propensity matched data between salvage HT and systemic CT for patients with persistent cervical cancer after RT/CCRT. Patients who did not undergo intracavity brachytherapy were excluded from the analysis. Figure A6. Survival comparison using propensity matched data between salvage HT and systemic CT for patients with persistent cervical cancer after RT/CCRT. Patients who had experienced crossover treatments were excluded from the analysis. Figure A7. Survival comparison using propensity matched data between salvage HT and systemic CT. Patients were divide into two groups according to whether imaging after definitive RT/CCRT revealed a residual lymph node tumor or not. Figure A8. Survival comparison using propensity matched data between salvage HT and systemic CT. Patients were divide into two groups according to whether imaging after definitive RT/CCRT revealed a residual cervical cancer or not. Figure A9. Survival comparison of patients in the systemic CT group, which was subdivided into 3 groups according to their response of each patient to chemotherapy . Table A1. Summary of treatments received. Table A2. Adverse events for systemic chemotherapy. Table A3. Evaluation of CT, MRI and PET-CT for persistent tumors after definitive RT/CCRT. Table A4. Sites of progressive disease after salvage HT or systemic CT. Table A5. Participating institutions and sample size.