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Inositol-Requiring Enzyme 1 (IRE-1) is an endoplasmic reticulum (ER) transmembrane protein with the ability to monitor ER luminal stress through its stress-sensing domain, subsequently initiating the UPR via its cytoplasmic kinase domain and RNase domain . Intriguingly, our recent, as-yet-unpublished research highlights that inhibiting the ERS-associated IRE-1 Pathway holds promise in alleviating PTB, indicating a potential connection between IRE-1 and PTB. Nonetheless, it remains uncertain whether LHX1 interacts with IRE-1 and actively participates in the UPR and the progression of PTB. A comprehensive exploration of these aspects is crucial to fully comprehend LHX1's role in PTB and ERS. | PMC467042_p3 | PMC467042 | Introduction | 4.262134 | biomedical | Study | [
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In the course of this study, we have identified that LHX1 binds to the IRE-1 promoter, subsequently promoting the transcriptional activation of IRE-1, thus contributing to PTB. These findings offer promising insights that may spark innovative ideas for the diagnosis and treatment of PTB. | PMC467042_p4 | PMC467042 | Introduction | 3.82982 | biomedical | Study | [
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Placenta samples were collected from a cohort of 30 parturients who experienced preterm births (PTB) and 30 parturients with normal-term pregnancies after the verbal consent. This sampling was conducted at the Fujian Maternity and Child Health Hospital, College of Clinical Medicine for Obstetrics & Gynecology, and Pediatrics, Fujian Medical University. Placental samples were acquired immediately following neonatal delivery and the rupture of placental membranes. Subsequently, these samples were promptly stored at a temperature of −80 °C within a freezer. This study had received prior approval from the Ethics Committee of the Fujian Maternity and Child Health Hospital, College of Clinical Medicine for Obstetrics & Gynecology, and Pediatrics, Fujian Medical University . Furthermore, each participant provided their informed consent to be part of the study. | PMC467042_p5 | PMC467042 | Tissue samples | 3.878528 | biomedical | Study | [
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The human placental trophoblast cell line, HTR8/SVneo, was procured from Procell (Wuhan, China). These cells were cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) and incubated at 37 °C in incubator with 5 % CO 2 . To simulate an ERS environment, the cells HTR8/Svneo were grown aseptically until they reached 80 % confluency and then treated with 5 μg/mL of tunicamycin (TM), for a duration of 24 h. | PMC467042_p6 | PMC467042 | Cell culture and treatment | 4.096024 | biomedical | Study | [
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The shRNA against LHX1 (sh-LHX1) with sequence of 5′- GATCCGAGGCTAAGTCTTGTGAACTCGAAAGTTCACAAGACTTAGCCTC -3′ 3′- GCTCCGATCTCGATTCAAGACTTGAATTCTCGAAGTGTTCTGAATCGGAG -5′and the negative control (sh-NC) 5′- GATCCGGAACAGGAGGCGCAACGAATCTCGAAAGATTCGTTGCGCCTCCTGTTCC -3′ 3′- GCTTGTCC TCTTCCGCGTTGCTTAGAAGCTTTCTAAGCAAGCGGAGGAACAA GG -5′ were purchased from Ribobio (Guangzhou, China). The full-length sequence of IRE-1: 5′-ATGCTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGC-3′) was inserted into pcDNA3.1 vector (Geenseed Biotech, Guangzhou, China) and the empty vector (5′- GTACGACTACGACTACGACTACGACTACGACTACGT-3′) served as NC. Cells were seeded in 6-well plates and grew to 80 % confluence. Lipofectamine 3000 was utilized for performing cell transfection. | PMC467042_p7 | PMC467042 | Cell transfection | 4.102444 | biomedical | Study | [
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Total RNA was extracted using TRIzol . Subsequently, the RNA underwent reverse transcription for cDNA synthesis, utilizing the Reverse Transcription Kit from Qiagen (Hilden, Germany). Following this, quantitative polymerase chain reaction (qPCR) was performed with the SYBR Green PCR Kit by Takara (Japan) on the StepOnePlus Real-Time PCR System by Applied Biosystems (USA). To quantify RNA expression levels, the 2 −ΔΔCt method was applied, with GAPDH serving as the control. | PMC467042_p8 | PMC467042 | RT-qPCR analysis | 4.113709 | biomedical | Study | [
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The primer sequences are presented in Table 1 . Table 1 Primers used in RT-qPCR. Table 1 Gene Forward sequence Reverse sequence XBP-1 5′-CTGTGAGCGAGTCCGAATC-3′ 5′-AGTCAATACCGCCAGAATGC-3′ LHX-1 5′- ATGGAGTACCTGAGGTGGCG -3′ 5′- CGCCACTCAGGTACTCCAT -3′ GRP78 5′-GCTTGTGGGTCTGGAGAAGA-3′ 5′-GACATCTTCTCCTGCGTCTG-3′ CHOP 5′-ACAGAGGAGGAGGAGGAGGA-3′ 5′-CTGTTCTCCTTCTCCTGCTG-3′ EIF2-α 5′-GCTGTTGGTGAAGGAGGAGA-3′ 5′-TGTCCAGTTCTGCTCCCTTC-3′ IRE-1 5′-TGCTTGGTGATGTGGAAAGG-3′ 5′-AGTTCTGGCTGGGTGTCTTT-3′ ATF6 5′-AGAGCGCTTCGAGAGGAAGT-3′ 5′-GGGTTTCTGTCCAGCTCCTT-3′ | PMC467042_p9 | PMC467042 | RT-qPCR analysis | 3.989682 | biomedical | Study | [
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] | [
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0.014766875654459,
0.0003941547474823892,
0.00020816699543502182
] | en | 0.999998 |
HTR8/SVneo cells were seeded on glass slides in 24-well plates. After fixing them with 4 % formaldehyde , the cells were treated with 0.1 % Triton X-100 . Subsequently, the cells were incubated with anti -LC3B at a dilution of 1:500 or anti -LHX1 at a dilution of 1:900 at 4 °C overnight. Following this, the cells were washed and incubated with a goat anti-rabbit secondary antibody for 2 h. DAPI was used to stain the cell nuclei and cells were finally observed using an Olympus microscope (Tokyo, Japan). | PMC467042_p10 | PMC467042 | Immunofluorescence (IF) | 4.118423 | biomedical | Study | [
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Cells (2 × 10 3 ) were seeded in 96-well plates and allowed to incubate for 24 h. Subsequently, they were treated with 10 μl of CCK-8 at various time points for an additional 2 h. The absorbance at 450 nm was measured using a microplate reader (Tecan Infinite, Mannedorf, Switzerland). | PMC467042_p11 | PMC467042 | Cell viability assays | 4.069868 | biomedical | Study | [
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] | en | 0.999999 |
Colony formation assays were conducted by seeding approximately 1000 cells into a 60-mm dish. Following a 14-day incubation in a uniform atmosphere with 5 % CO2 at 37 °C, the resultant clones were fixed using methyl alcohol and stained with crystal violet for 30 min. Afterward, they were washed with ddH2O and manually counted under a microscope. | PMC467042_p12 | PMC467042 | Colony formation assays | 4.098639 | biomedical | Study | [
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Migratory and invasive capabilities of HTR8/SVneo cells were evaluated using Transwell chambers with or without Matrigel . Cells were seeded in the upper chamber of the Transwell with serum-free RPMI medium, while the lower chamber was filled with complete culture medium. After 24 h, cells that had migrated to the lower chamber were fixed with 4 % paraformaldehyde , stained with crystal violet (Sigma #548-62-9), and observed under a microscope (Olympus). | PMC467042_p13 | PMC467042 | Transwell assay | 4.109859 | biomedical | Study | [
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Cell apoptosis in HTR8/SVneo cells was assessed using the Annexin V/Dead Cell Apoptosis kit . After transfection, cells were double-stained with 5 μl of Annexin V-FITC and 5 μl of propidium iodide (Thermo Fisher, #BMS500PI) in the dark for 15 min. Subsequently, a FACS Calibur flow cytometer (BD Biosciences, USA) in conjunction with FlowJo software was employed to measure the cell apoptosis rate. | PMC467042_p14 | PMC467042 | Flow cytometry | 4.095444 | biomedical | Study | [
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Total proteins were extracted using RIPA buffer . After electrophoresis on SDS-PAGE, the proteins were transferred onto PVDF membranes and blocked with 5 % nonfat milk. Subsequently, they were incubated with primary antibodies, including LHX-1 , GAPDH , LC3 I & II (Millipore Sigma#ABC929, 1:300), p62 , ATG5 , Beclin 1 , IRE-1 , CHOP , ATF6 , GRP78 , EIF2-α , and p-EIF2-α , at 4 °C overnight. This was followed by incubation with secondary antibodies for 2 h. Protein bands were detected using the ECL kit (Millipore, Billerica, MA, USA) and analyzed using ImageJ. | PMC467042_p15 | PMC467042 | Western blot | 4.093274 | biomedical | Study | [
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Cells were initially cross-linked using 1 % formaldehyde. Subsequently, lysis buffer was introduced to the treated cells, and the chromatin was fragmented into DNA fragments of 150–900 bp through sonication. Following this, anti -LHX1 or a control anti -IgG was included in the sonicated mixtures for incubation. The precipitated complexes were then subjected to washing and reverse cross-linking. After purification, the isolated DNA was analyzed using RT-qPCR. | PMC467042_p16 | PMC467042 | ChIP assay | 4.137169 | biomedical | Study | [
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] | [
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Luciferase reporter assay was carried out to assess the impact of LHX1 binding on the IRE-1 promoter's regulatory activity. Briefly, the wild-type (WT) or mutated (Mut) LHX1 binding sites within the IRE-1 promoter region were sub-cloned into the luciferase reporter vector pGL3 (Promega, Madison, WI). Subsequently, cells were co-transfected with the reporter vectors and the specified plasmids. After a 48-h incubation period, the Luciferase Reporter Assay System (Promega) was utilized to conduct the luciferase activity analysis. | PMC467042_p17 | PMC467042 | Luciferase reporter assay | 4.103324 | biomedical | Study | [
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We induced a PTB mouse model using the ERS inducer, TM. Nakashima et al. showed that intraperitoneal injection of TM successfully initiated a premature increase in myometrial gap junction protein alpha 1 levels, elevated contractile responsiveness, and advanced the onset of preterm labor in mice. These effects were attributed to the induction of an exaggerated UPR . Prior to conducting this experiment, we obtained the necessary ethical clearance from the Ethics Committee of the Fujian Maternity and Child Health Hospital College of Clinical Medicine for Obstetrics & Gynecology and Pediatrics, Fujian Medical University . For this study, timed pregnant CD1 mice were procured from the Fujian Maternity and Child Health Hospital College of Clinical Medicine for Obstetrics & Gynecology and Pediatrics, Fujian Medical University, and they were raised in a controlled SPF environment. On E13, pregnant mice underwent one of the following treatments: a vehicle injection (sh-NC, 5′-GACGTTCAGGCTTGCACTTGA-3′), an intraperitoneal injection of 0.2 mg/kg TM, or a combined injection of TM + Sh-LHX1, with the objective of delivering small hairpin RNA (shRNA) 5'-(GCTGCAAGGCTGACTTACGTT-3′) targeting the LHX1 gene. These treatments were administered over a 24-h period. Mice in the TM + Sh-LHX1 group received an intravenous injection of 500 μg of shRNA treatment in 0.5 mL of PBS via the tail vein 1 h after the administration of TM. Subsequently, the mice were euthanized, or they were monitored until they reached full term. Placenta samples were collected, and they were promptly stored at −80 °C for subsequent analysis. | PMC467042_p18 | PMC467042 | PTB mouse model | 4.109494 | biomedical | Study | [
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Placenta samples were sectioned into 5-μm slices, and the slides were passed through three changes of xylene followed by graded ethanol for deparaffinization and rehydration. Antigen retrieval from the tissues was performed by heating them in a sodium citrate buffer in an oven. Subsequently, the slides were treated with 3 % hydrogen peroxide for 15 min and then blocked with goat serum. The tissue sections were then incubated with primary antibodies against LHX1 and LC3 (Millipore Sigma#ABC929, 1:100) overnight at 4 °C. Following this, they were incubated with a goat anti-rabbit secondary antibody for 2 h. After thorough washing with phosphate buffer saline, the slides were treated with DAB substrate for 10 min and counterstained with hematoxylin . Finally, the slides were washed again and mounted with mounting media before being observed using a microscope (Olympus, Japan). | PMC467042_p19 | PMC467042 | Immunohistochemistry (IHC) | 4.168495 | biomedical | Study | [
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The tissue slides underwent dewaxing with xylene , rehydration through varying ethanol concentrations , and a subsequent permeation in 0.1 % Triton X-100 in PBS for 5 min. Following this, Equilibration Buffer (100 μl) was applied to the slides at room temperature for 10 min. Staining was executed using the TUNEL assay kit, In Situ Cell Death Detection Kit (Roche, Basel, Switzerland), as per the provided instructions. Apoptotic cells were observed at a magnification of × 100 using an Olympus microscope (Tokyo, Japan). To quantify apoptotic cells, eight randomly chosen fields were selected for manual counting, and image analysis was conducted using Motic Med 6.0 software (Xiamen Motic Software Engineering Co., Ltd., Xiamen, China). The apoptosis index was then determined by dividing the number of apoptotic cells by the total number of cells and multiplying by 100 %. | PMC467042_p20 | PMC467042 | TUNEL assay | 4.140763 | biomedical | Study | [
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] | [
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The data were reported as means ± standard deviation (SD) derived from three independent replicates. Statistical analysis was conducted using GraphPad Prism 6 software, employing either a Student's t-test or a one-way analysis of variance (ANOVA). A significance threshold of P < 0.05 was applied to ascertain statistical significance. | PMC467042_p21 | PMC467042 | Statistical analyses | 3.300248 | biomedical | Study | [
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] | [
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In our initial investigation, we assessed LHX1 expression in placentas affected by PTB. The results from RT-qPCR revealed a significant upregulation of LHX1 expression in PTB placenta compared to normal full-term placenta . Furthermore, IHC analysis corroborated this heightened expression of LHX1 in PTB placenta . Western blot analysis provided additional confirmation of elevated LHX1 protein levels in PTB placenta . Subsequently, we established a cellular model of ERS injury in PTB by treating human trophoblast cells with tunicamycin (TM) and examined LHX1 expression in these cells. Both LHX1 mRNA and protein levels exhibited marked increases upon TM induction . Additionally, IF results demonstrated strong fluorescence intensity of LHX1 in TM-induced cells . Overall, our results demonstrated that increased expression of LHX1 in PTB might play a role in regulating PTB development. Fig. 1 Elevated LHX1 Expression in PTB Placenta. (A) RT-qPCR analysis comparing LHX1 expression in normal term placenta (n = 30) and PTB placenta (n = 30), illustrating a substantial upregulation of LHX1 in PTB samples. (B) IHC staining highlights increased LHX1 expression in the representative sections of the placenta of control group (n = 3) and PTB group (n = 3) providing visual confirmation of heightened LHX1 levels in PTB tissues. (C) Western blot results indicating elevated LHX1 protein levels in the placenta of control group (n = 3) and PTB group (n = 3). (D-E) RT-qPCR and Western blot analysis of LHX1 expression in control and TM-treated HTR8/Svneo cells (n = 3 per group). Both mRNA and protein levels of LHX1 exhibit marked increases upon TM induction, indicating ERS-induced upregulation of LHX1. (F) IF assay demonstrates increased LHX1 expression in control and TM-treated HTR8/Svneo cells (n = 3 per group), further supporting the observed increase in LHX1 expression. ***P < 0.001 vs control. Fig. 1 | PMC467042_p22 | PMC467042 | LHX1 expresses at a high level in PTB placenta | 4.198274 | biomedical | Study | [
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We next treated HTR8/Svneo cells with TM, and the impact of TM on cellular behaviors and ERS-related markers was evaluated. Our findings demonstrate that TM exerted significant effects on HTR8/Svneo cells, leading to the suppression of cell viability, proliferation, migration, and invasion. Moreover, TM treatment resulted in the promotion of autophagy in HTR8/Svneo cells, accompanied by an induction of ERS markers, including XBP-1, CHOP, AF6, and GRP78 . | PMC467042_p23 | PMC467042 | LHX1 promotes autophagy and apoptosis of trophoblast cells | 4.142928 | biomedical | Study | [
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We then knocked down LHX1 expression in cells to investigate its impact on cellular behavior. RT-qPCR results showed the transfection efficiency of sh-LHX1 in HTR8/Svneo cells induced with TM . Interestingly, the knockdown of LHX1 significantly increased the viability and proliferation of cells treated with TM compared to cells induced with TM but transfected with sh-NC . Transwell assays confirmed that silencing LHX1 enhanced cell migratory and invasive capabilities under TM stimulation . Subsequently, we observed a significant decrease in the rate of cell apoptosis in the TM + sh-LHX1 group . We next examined the impact of LHX1 silencing on autophagy by monitoring levels of autophagy markers. Our results indicated a reduction in the ratio of LC3II/LC3I, decreased levels of ATG5 and Beclin-1, and increased levels of P62 in cells induced with TM due to LHX1 silencing, suggesting the inhibition of autophagy . These results were further confirmed by the diminished fluorescence intensity of the autophagy marker LC3 in TM + sh-LHX1-induced cells . Therefore, our findings suggest that under TM stimulation, LHX1 restrains the migratory and invasive capabilities of trophoblast cells, while promoting autophagy and apoptosis however all of these effects were reversed in absence of LHX1. Fig. 2 LHX1 promotes autophagy and apoptosis of trophoblast cells. (A) RT-qPCR results demonstrating the transfection efficiency of sh-LHX1 in HTR8/Svneo cells induced with TM (n = 3). (B–C) Cell viability and proliferation of HTR8/Svneo cells were assessed by CCK-8 and colony formation assays after treatment with TM and transfection with sh-NC or sh-LHX1 (n = 3 per group). (D–E) Evaluation of cell migratory and invasive capabilities using transwell assays (n = 3). Flow cytometry analysis was performed to determine the cell apoptotic rate after treatment with TM and transfection with sh-NC or sh-LHX1 (n = 3 per group). (G) Western blot results depicting protein levels of LC3, P62, ATG5, and Beclin-1 in HTR8/Svneo cells after treatment with TM and transfection with sh-NC or sh-LHX1 (n = 3 per group). (H) Immunofluorescence (IF) assay for assessing LC3 1 in HTR8/Svneo cells after treatment with TM and transfection with sh-NC or sh-LHX1 (n = 3 per group). ***P < 0.001 vs sh-NC. Fig. 2 | PMC467042_p24 | PMC467042 | LHX1 promotes autophagy and apoptosis of trophoblast cells | 4.189095 | biomedical | Study | [
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IRE is the ER transmembrane receptor and IRE-1/XBP1/CHOP signaling pathway plays a crucial role in maintaining ER stability and is a key component in ERS. This prompted us to investigate the relationship between IRE-1 and LHX1. Interestingly, Pearson's correlation analysis indicated a positive regulatory connection between LHX1 and IRE-1 . This led us to hypothesize that LHX1 might be involved in the development of preterm birth (PTB) by influencing the IRE-1/XBP1/CHOP pathway. Fig. 3 LHX1 modulates the IRE-1/XBP1/CHOP signaling pathway . (A) Assessment of the expression correlation between IRE-1 and LHX1 in 30 preterm birth (PTB) placenta samples. The analysis, performed using Pearson's correlation, indicates a significant correlation , suggesting a potential functional association between IRE-1 and LHX1. (B–C) RT-qPCR and Western blot analysis results showing the mRNA and protein expression levels of IRE-1, XBP-1, and CHOP in HTR8/Svneo cells from the TM group, TM + sh-NC group, and TM + sh-LHX1 group (n = 3 per group). (D–E) RT-qPCR and Western blot analysis results illustrating the mRNA and protein expression levels of ATF6, GRP78, and eIF2α in HTR8/Svneo cells of TM group, TM + sh-NC, and TM + sh-LHX1 groups (n = 3 per group). ***P < 0.001 vs TM + sh-NC. Fig. 3 | PMC467042_p25 | PMC467042 | LHX1 regulates the IRE-1/XBP1/CHOP signaling pathway | 4.131771 | biomedical | Study | [
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To test this hypothesis, we conducted experiments to observe mRNA and protein levels of IRE-1, XBP1, and CHOP and ERS related genes. Our results revealed a significant decrease in IRE-1, XBP1, and CHOP mRNA and protein levels when LHX1 was knocked down in TM-induced HTR8/Svneo cells . Furthermore, we observed that the deficiency of LHX1 led to a reduction in the mRNA levels of ERS-related genes ATF6, GRP78, and eIF2α . Western blot analysis confirmed a notable decrease in ATF6, GRP78, and p-eIF2α levels in response to LHX1 deficiency . These findings provide strong evidence that LHX1 activates the IRE-1/XBP1/CHOP signaling pathway, thus contributing to PTB development. | PMC467042_p26 | PMC467042 | LHX1 regulates the IRE-1/XBP1/CHOP signaling pathway | 4.181453 | biomedical | Study | [
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We next aimed to investigate the physical interaction between LHX1 and IRE-1. We thus utilized the JASPAR database to predict the binding sites of LHX1 and IRE-1 . To provide a more detailed insight, we further depicted the sequence diagram of the LHX1 motif and the binding sites between LHX1 and the IRE-1 promoter . To experimentally validate this interaction, we conducted a ChIP assay, and the results clearly demonstrate that the enrichment of the IRE-1 promoter was significantly elevated in the LHX1 group when compared to the IgG control in HTR8/Svneo cells and human PTB placentas . Moreover, we assessed the functional consequences of LHX1 binding to the IRE-1 promoter. Notably, the luciferase activity of the wild-type IRE-1 promoter was significantly enhanced in response to LHX1 overexpression, whereas this effect was not observed with IRE-1 mutants, as demonstrated in both 293 T and HTR8/Svneo cells . Taken together, these findings provide strong evidence that LHX1 specifically targets the IRE-1 promoter and exerts a transcriptional activating effect on its expression. Fig. 4 LHX1 transcriptionally activates IRE-1 expression. (A) Predicted binding sites of LHX1 on the IRE-1 promoter obtained from the JASPAR database, laying the foundation for investigating the physical interaction between LHX1 and IRE-1. (B) DNA motif of LHX1 obtained from the JASPAR database, providing a sequence diagram for a detailed insight into the binding sites. (C) Schematic representation illustrating the interaction between LHX1 and the IRE-1 promoter, highlighting the predicted binding sites. (D) ChIP assay results demonstrating the binding of LHX1 to the IRE-1 promoter in 293T, HTR8/Svneo cells, and human PTB placentas (n = 3 per group). Anti -IgG antibody served as the negative control for the anti -LHX1 antibody. (E) Luciferase reporter assays confirming the transcriptional activation effect of LHX1 on the IRE-1 Promoter in 293T and HTR8/Svneo cells (n = 3 per group). Cells were co-transfected with pcDNA3.1 vector expressing LHX1 and pGL3 vector expressing wild type or mutated IRE-1 promoter. **P < 0.01, ***P < 0.001 vs IgG or control. Fig. 4 | PMC467042_p27 | PMC467042 | Transcriptional activation of IRE-1 expression by LHX1 | 4.233555 | biomedical | Study | [
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Our next goal was to examine how LHX1, and IRE-1 impact the regulation of trophoblast cell functions. We have shown in previous results that silencing LHX1 led to an enhancement in cell proliferation and viability. However, these effects were significantly reversed when IRE-1 was overexpressed . Furthermore, transwell assay revealed that IRE-1 overexpression effectively countered the migratory and invasive capabilities caused by LHX1 knockdown in TM-induced HTR8/Svneo cells . Moreover, when assessing the apoptotic rate of cells, we observed that inhibitory effect on apoptosis due to LHX1 deficiency was rescued or even enhanced when IRE-1 was overexpressed . Additionally, our study examined that IRE-1 upregulation reversed the inhibitory effect of LHX1 deficiency on protein levels of LC3II/LC3I, ATG5 level, Beclin-1 level, and reversed the promoting effect on P62 level . Moreover, we delved into the activity of the IRE-1/XBP1/CHOP pathway, which was found to be suppressed by LHX1 silencing as shown in previous results. However, when IRE-1 was upregulated, it effectively reactivated this pathway . In summary, our results collectively highlight that LHX1 plays a crucial role in regulating the migration, invasion, apoptosis, and autophagy of trophoblast cells. It accomplishes this by upregulating IRE-1, thereby underscoring the intricate interplay between these two factors in the context of trophoblast cell behavior. Fig. 5 LHX1 Upregulation Modulates Trophoblast Cell Behaviors Through IRE-1. (A–B) CCK-8 and MTT assays were employed to evaluate cell viability in TM-induced HTR8/Svneo cells across various groups, including the sh-NC group, the sh-LHX1 group, the sh-LHX1+Vector group, and the sh-LHX1+IRE-1 group (n = 3 per group). (C–D) Cell migratory and invasive capabilities of sh-NC group, sh-LHX1 group, sh-LHX1+Vector group, and sh-LHX1+IRE-1 groups were assessed through Transwell assays (n = 3 per group). (E) Cell apoptotic rates of sh-NC group, sh-LHX1 group, sh-LHX1+Vector group, and sh-LHX1+IRE-1 groups were determined by flow cytometry analysis (n = 3 per group). (F–H) Western blot analysis was performed to examine the protein levels of LC3, P62, ATG5, Beclin-1, IRE-1, XBP-1, and CHOP in HTR8/Svneo cells of sh-NC group, sh-LHX1 group, sh-LHX1+Vector group, and sh-LHX1+IRE-1 groups (n = 3 per group). **P < 0.01, ***P < 0.001 compared with the sh-NC group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the sh-LHX1+Vector group. Fig. 5 | PMC467042_p28 | PMC467042 | LHX1 regulates trophoblast cell behaviors by upregulating IRE-1 | 4.234623 | biomedical | Study | [
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We established a PTB mouse model to confirm the in vivo role of LHX1 in PTB development. The mice were divided into three groups (n = 5/group): a control group, a group subjected to TM treatment to induce ERS, and a group treated with both TM and Sh-LHX1 to simultaneously reduce LHX1 gene expression. Mice subjected to TM treatment had a shortened pregnancy period, approximately 16 days, indicative of PTB development. In contrast, mice receiving TM-sh-LHX1 treatment had a normal pregnancy duration, generally lasting 20 days . Furthermore, the live birth rate in the TM-treated mice was lower than that of the control group, while the TM-sh-LHX1-treated mice exhibited a higher live birth rate than the TM-treated group . To investigate the impact on placental tissue, we conducted TUNEL assays to assess apoptosis. We observed a notable increase in the rate of apoptosis in the TM group, while the TM-sh-LHX1-treated mice exhibited a reduction in the high apoptosis rate induced by TM . Additionally, to assess the effects on autophagy, we examined the expression of LC3. IHC confirmed enhanced LC3 expression in the placenta of the TM group. However, LHX1 knockdown led to reduced LC3 expression, indicating the inhibition of autophagy, consistent with our in vitro findings . Furthermore, we investigated the expression of LHX1, IRE-1, XBP-1, CHOP, AF6, and GRP78 at both mRNA and protein levels. TM induction resulted in elevated mRNA expression of these factors, while LHX1 knockdown had the opposite effect, aligning with our in vitro results . Furthermore, our analysis revealed that LHX1 significantly bound to the IRE-1 promoter in PTB placentas, demonstrating a potential regulatory role . Additionally, silence of LHX1 reduced IRE-1, XBP-1, CHOP, AF6, and GRP78 protein levels in placentas of PTB mice . These findings collectively suggest that silencing LHX1 can ameliorate the PTB phenotype in mice. Fig. 6 LHX1 knockdown reduces PTB symptom in mice. (A) Statistical analysis of the pregnancy duration in mice across three groups: the control group, the TM group, and the TM + sh-LHX1 group (n = 5 per group). (B) Assessment of the live birth rate in control group, TM group, and TM + sh-LHX1 group (n = 5 per group). (C) Measurement of the rate of cell apoptosis in placental tissues of control group, TM group, and TM + sh-LHX1 group using the TUNEL assay (n = 5 per group). (D) Visualization of LC3-positive regions in placental tissues of control group, TM group, and TM + sh-LHX1 group through IHC (n = 3 per group). (E) Evaluation of mRNA levels of LHX1, IRE-1, XBP-1, CHOP, AF6, and GRP78 in placental tissues of control group, TM group, and TM + sh-LHX1 group, conducted via RT-qPCR (n = 3 per group). (F) A ChIP assay was used to assess the binding of LHX1 on IRE-1 promoter in placenta tissues of PTB mice. (G) Western blot was performed to assess protein levels of LHX1, IRE-1, XBP-1, CHOP, AF6, and GRP78 in placental tissues of control group, TM group, and TM + sh-LHX1 group (n = 3 per group). **P < 0.01, ***P < 0.001 compared with the control group; ###P < 0.001 compared with the TM group. Fig. 6 | PMC467042_p29 | PMC467042 | LHX1 knockdown alleviates PTB symptom in mice | 4.252094 | biomedical | Study | [
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This study investigates the association between ERS, specifically mediated by the UPR, and PTB. In the development of the placenta, invasive extravillous trophoblasts (EVTs) play a critical role by facilitating the production of pregnancy hormones and performing key functions, including fetal immune defense . The inadequacy of EVT invasion can lead to acute obstetrical complications, including PTB . Our results reveal a significant upregulation of LHX1 in PTB placenta, both at the mRNA and protein levels. The induction of ERS in trophoblast cells further corroborated the marked increase in LHX1 expression under the influence of TM, highlighting its potential role in regulating PTB development. In exploring the functional implications of LHX1, our study demonstrates its impact on trophoblast cell behaviors, including migration, invasion, apoptosis, and autophagy, particularly when influenced by TM stimulation. LHX1 appears to exert these effects by modulating the IRE-1/XBP1/CHOP signaling pathway. The intricate relationship between LHX1 and IRE-1 is further supported by our findings that LHX1 transcriptionally activates IRE-1 expression, establishing a novel regulatory axis in the context of PTB. Furthermore, significance of LHX1 extends beyond the trophoblast domain, marking a pivotal presence during the emergence of the anterior visceral endoderm in utero . Our in vivo findings align with our in vitro data, demonstrating that silencing LHX1 alleviates PTB symptoms, normalizing pregnancy duration and live birth rates while reducing apoptosis and modulating autophagy in placental tissues. These comprehensive results collectively underscore the indispensable role of LHX1 in placental development and its potential impact on the intricate pathways leading to PTB. | PMC467042_p30 | PMC467042 | Discussion | 4.615743 | biomedical | Study | [
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Autophagy represents a highly conserved degradation pathway to maintain cellular equilibrium and plays a pivotal role in embryonic and placental development . Augmented autophagy is associated with the rupture of the amniotic membrane that envelops the fetuses during pregnancy . Variations in placental autophagy features have been observed in PTB . Furthermore, autophagy can be induced by the UPR, and key ER proteins involved in modulating autophagy encompass PERK, IRE-1, and ATF6 . LC3 is commonly employed as an autophagosome marker. Other frequently used genes related to autophagy encompass ATG5, Beclin-1, and P62. P62 serves as an autophagy adaptor protein, facilitating the selective degradation of proteins [ , , ]. In our study, we have established that the knockdown of LHX1 inhibits cell apoptosis, reduces the levels of LC3, ATG5, and Beclin-1, and increases P62 levels. These findings suggest that LHX1 deficiency suppresses autophagy in TM-stimulated HTR8/SVneo cells. | PMC467042_p31 | PMC467042 | Discussion | 4.254874 | biomedical | Study | [
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IRE-1 is an ER luminal stress-sensing domain to monitor conditions and activates the UPR . When the ER experiences an accumulation of unfolded or misfolded proteins, IRE-1 dissociates from GRP78, resulting in the splicing of XBP1 mRNA to produce XBP1s . Notably, XBP1s exhibits the remarkable ability to bind to ERS response elements found in the promoters of numerous UPR target genes. This, in turn, aids in folding and degrading proteins, ultimately promoting ER adaptation and cellular protection . Our investigation revealed a novel facet of IRE-1 function, wherein it transcriptionally activates LHX1 by binding to its promoter specifically in TM-stimulated HTR8/Svneo cells and placental tissues. The significance of IRE-1 in placental development and embryonic viability has been well-established . Intriguingly, experiments involving the overexpression of IRE-1 showcased its capability to counteract the effects of LHX1 depletion. Specifically, it mitigated the impact on cell migration, invasion, apoptosis, and autophagy in HTR8/Svneo cells. These findings provide compelling evidence for the substantial contribution of IRE-1 in ERS-induced PTB, shedding light on its pivotal role in the intricate molecular pathways governing placental dynamics. Notably, under severe and prolonged stress, ERS can trigger the apoptotic pathway . This UPR-mediated apoptosis pathway is a novel one, and it involves the activation of CHOP , a transcription factor that induces cell apoptosis and is regulated by XBP1 . Activation of the IRE-1/XBP1/CHOP pathway has been demonstrated to promote cell apoptosis and inflammatory reactions, leading to the disruption of the intestinal barrier . Additionally, reports indicate elevated expression levels of GRP78, IRE-1, and XBP1 in PTB fetal membranes, which are associated with embryonic lethality and inhibiting ERS may hold promise as a potential therapeutic strategy for addressing PTB . In our study, LHX1 knockdown significantly reduces the levels of IRE-1, XBP1, and CHOP in TM-induced HTR8/Svneo cells. Additionally, mRNA levels of ERS-related genes ATF6, GRP78, and eIF2α are inhibited by the deficiency of LHX1. Consequently, we can confirm that LHX1 activates the IRE-1/XBP1/CHOP signaling pathway in PTB. In summary, our study provides comprehensive insights into the regulatory roles of LHX1 in PTB, unraveling its connections to ERS and the IRE-1/XBP1/CHOP pathway. These findings open avenues for further research and potential therapeutic interventions aimed at mitigating the impact of PTB. | PMC467042_p32 | PMC467042 | Discussion | 4.754832 | biomedical | Study | [
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The experimental protocol was established according to the ethical guidelines of the Helsinki Declaration and was approved by Ethics Committee of the Fujian Maternity and Child Health Hospital, College of Clinical Medicine for Obstetrics & Gynecology, and Pediatrics, Fujian Medical University . The animal studies received approval from the Ethics Committee of the Fujian Maternity and Child Health Hospital College of Clinical Medicine for Obstetrics & Gynecology and Pediatrics, Fujian Medical University . | PMC467042_p33 | PMC467042 | Ethics approval and consent to participate | 0.924197 | biomedical | Other | [
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This work was supported by the Joint Funds for the innovation of science and Technology , Fujian province . | PMC467042_p34 | PMC467042 | Funding | 0.994892 | other | Other | [
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The data that support the findings of this study are available from the corresponding author upon reasonable request. | PMC467042_p35 | PMC467042 | Data availability statement | 0.925836 | other | Other | [
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Liyin Qiu: Writing – original draft, Software, Project administration, Methodology. Zhaozhen Liu: Writing – review & editing, Methodology, Formal analysis, Data curation. Shouzhen Chen: Writing – review & editing, Project administration, Methodology, Data curation. Yiting Wu: Writing – review & editing, Software, Formal analysis. Jianying Yan: Resources, Investigation, Funding acquisition. | PMC467042_p36 | PMC467042 | CRediT authorship contribution statement | 0.991829 | other | Other | [
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | PMC467042_p37 | PMC467042 | Declaration of competing interest | 0.981821 | other | Other | [
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Neonatal seizures are the most common neurological manifestation of brain injury in the neonatal period. 1 Affected neonates die or survive with long-term neuro-disability and epilepsy. 2 The burden of neonatal seizures in low- and middle-income countries (LMIC) is 10–30 times higher than in high-income countries. 3 , 4 | 39021480_p0 | 39021480 | Introduction | 3.432112 | biomedical | Review | [
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Clinical diagnosis of neonatal seizures is unreliable and subjective. Non seizures movements may be mis-interpreted as seizures leading to unnecessary treatment. Conversely, many electrographic seizures may not have clinical manifestations. 5 , 6 , 7 Hence, medical regulatory bodies recommend that in clinical trials evaluating antiseizure medication (ASM), seizure freedom should be examined using EEG. 8 , 9 , 10 | 39021480_p1 | 39021480 | Introduction | 3.695336 | biomedical | Other | [
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As an ASM, levetiracetam has several advantages, particularly in LMIC due to its safety profile, minimal sedation, lack of respiratory suppression, ease of administration, favorable pharmacokinetics and potential neuroprotection. 11 , 12 Pooled data from several small single centre open label randomised controlled trials from LMIC suggest levetiracetam has similar efficacy to phenobarbital in clinical seizure cessation (70% versus 56%). 13 , 14 , 15 However, the efficacy of levetiracetam was much lower than phenobarbital (28% versus 80%) in a well-designed multicenter phase IIb randomised controlled trial where EEG was used to assess seizure cessation. 16 | 39021480_p2 | 39021480 | Introduction | 4.049566 | biomedical | Study | [
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We evaluated the efficacy of sequential levetiracetam and phenytoin administration in terminating neonatal seizures unresponsive to phenobarbital using continuous EEG monitoring in South India. | 39021480_p3 | 39021480 | Introduction | 3.689148 | biomedical | Study | [
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We conducted a prospective multicenter observational study across three tertiary care public sector teaching hospitals in India (Karnataka Institute of Medical Sciences, Hubballi; Bangalore Medical College and Research Institute, Bengaluru; and Government Medical College, Kozhikode) between 20 June 2020 and 31 July 2022. | 39021480_p4 | 39021480 | Study design and participants | 3.015239 | biomedical | Study | [
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Neonates born at the recruiting hospital were defined as inborn, and the neonates born at other health care facilities or at home were defined as outborns. All inborn neonates born at or after 35 weeks of gestation and admitted to neonatal unit with encephalopathy or clinical seizures within 72 h after birth were screened for eligibility. Upon recognition of clinical or electrographic seizures, phenobarbital was administered as the first line ASM (a total of 30–40 mg/kg in 2 doses) if seizures continued after metabolic corrections. | 39021480_p5 | 39021480 | Study design and participants | 3.982419 | biomedical | Study | [
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We included all neonates who had continued seizures on EEG after 30 min of phenobarbital administration requiring additional ASM. The following neonates were excluded 1) outborn neonates 2) neonates who received second line ASM without EEG monitoring 3) transient metabolic disorders who responded to metabolic corrections and inborn errors of metabolism. | 39021480_p6 | 39021480 | Study design and participants | 3.370511 | biomedical | Study | [
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The study was approved by research ethics committees at Imperial College, London and the participating sites, and all parents provided written informed consent. | 39021480_p7 | 39021480 | Study design and participants | 1.027312 | biomedical | Other | [
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Prior to the study, the PREVENT (Prevention of Epilepsy by Reducing Neonatal Encephalopathy) research consortium was set up between Imperial College London, University College London, Oxford University and the three recruiting sites in India. A team of 6 neonatal neurology fellows, 6 neonatal research nurses, and 7 EEG technicians were appointed at the study sites in India and were trained and certified in various aspects of the study protocol including structured neurological assessment (modified Sarnat stage), EEG acquisition and interpretation. All sites were provided with Neurosoft-Neuron-Spectrum 4-P video EEG machines (Neurosoft LLC, Ivanovo, Russia), and were read with Neuron spectrum software version 2.0.22.1. The montage was based on the international 10–20 system modified for neonates with 13 electrodes. The video was recorded time-locked with the EEG and additional polygraphic channels included electrocardiography (ECG), respiratory effort, and bilateral surface electromyographic recordings (deltoid). | 39021480_p8 | 39021480 | Procedures | 4.059096 | biomedical | Study | [
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EEG recordings were commenced between 6 and 24 h after birth in neonates admitted with HIE ensuring peak seizure occurrence was captured. In neonates admitted with suspected seizures or encephalopathy unrelated to hypoxic ischaemic encephalopathy (HIE), EEG was started soon after admission. In all neonates, EEG was continued for at least 4 h if the recording was normal and up to 24 h if seizures were noted. The EEG reporting was undertaken by specialist neonatal neurology fellows (VK, VU and HV) at each site in real time under the supervision of an expert clinical neurophysiologist (RP) using a cloud-based real time EEG review system. All seizures were verified by two independent reviewers. | 39021480_p9 | 39021480 | Procedures | 3.934342 | biomedical | Study | [
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A seizure management protocol based on current evidence-based recommendations for LMIC 17 was standardized across the sites and involved a step wise escalation starting with phenobarbital, followed by levetiracetam, then phenytoin, and finally midazolam. Neonates included in the study received levetiracetam (20 mg/kg) initially as a short infusion or slow iv push over 10–20 min and the dose was repeated if seizures persisted to achieve a maximal dose of 40 mg/kg. If seizures persisted despite maximal dose of levetiracetam, phenytoin (20 mg/kg) was administered as an infusion over 20–30 min and the dose was repeated (total 40 mg/kg) if seizures persisted. In between each infusion, a time gap of 10–20 min was given for the ASM to act unless the infant was in status epilepticus, where drug doses were escalated more rapidly. | 39021480_p10 | 39021480 | Procedures | 4.049573 | biomedical | Study | [
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Seizures were grouped into clinical events (no ictal EEG available), electrographic-only (EEG seizures without clinical manifestations), or electro-clinical seizures (EEG seizures with a clinical correlate). 4 Diagnostic certainty of seizures was documented as defined by the Brighton Collaboration Neonatal Seizures Working Group. 4 , 18 Level 1 included seizures confirmed with EEG, level 2 included clinical focal clonic or tonic seizures or seizures on amplitude integrated EEG (aEEG), and level 3 included other clinical events suggestive of epileptic seizures other than focal clonic or tonic. The clinical events not meeting case definitions (level 4) and those not having an EEG correlate (level 5) were taken as non-seizure events. 4 , 18 Seizure semiology was classified according to the ILAE seizure classification. 18 The EEG background was grouped according to the following criteria: normal (continuous activity with age appropriate graphoelements and well defined sleep wake cycling); mildly abnormal (continuous activity with mild asymmetry, voltage depression and/or poorly defined sleep wake cycle); moderately abnormal (discontinuous activity with interburst intervals less than 10 s, absent sleep wake cycles and clear asymmetry or asynchrony); severe (discontinuous activity with prolonged interburst intervals more than 10 s, severe attenuation, burst suppression and isoelectric patterns); or undetermined (difficult to assess background due to status epilepticus or excessive artefacts). 19 Status epilepticus was defined as a seizure burden of 30 min per hour or more in at least one 1-h epoch of EEG recording. 20 Seizure burden (minutes/hour) was defined as the total duration of ictal discharges (minutes) divided by the total duration of EEG (hours). | 39021480_p11 | 39021480 | Procedures | 4.276261 | biomedical | Study | [
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All neonates had detailed clinical assessments, electrolyte and blood sugar measurements, infection screening and magnetic resonance imaging prior to hospital discharge. Additional metabolic and genetic investigations were performed as clinically indicated. | 39021480_p12 | 39021480 | Procedures | 2.304858 | biomedical | Study | [
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The primary outcome was the onset of seizure freedom within 40 min from of the start of the initial dose of levetiracetam or phenytoin infusion. Seizure freedom was defined as a complete absence of seizures on continuous EEG monitoring for at least 60 min from the end of the last seizure without the need for any additional ASM. | 39021480_p13 | 39021480 | Outcomes | 3.981099 | biomedical | Study | [
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The efficacy of levetiracetam and phenytoin reported as proportions of neonates achieving seizure freedom along with their Clopper Pearson exact 95% confidence limits. To show the time to achieve seizure freedom (endpoint), Kaplan–Meier survival plots are plotted separately for neonates who received levetiracetam as second line ASM, and for neonates who received levetiracetam as well as third line ASM, phenytoin. Data were analysed using SPSS software, version 29.0. | 39021480_p14 | 39021480 | Statistical analysis | 3.988172 | biomedical | Study | [
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This research was funded by the National Institute for Health and Care Research (NIHR) Research and Innovation for Global Health Transformation using UK aid from the UK Government to support global health research. The views expressed in this publication are those of the author(s) and not necessarily those of the NIHR or the UK government. The study funders had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit the paper for publication. | 39021480_p15 | 39021480 | Role of funding source | 1.094119 | other | Other | [
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During the 2-year study period, a total of 1027 neonates born at or after 35 weeks were admitted to the neonatal intensive care unit (NICU) with encephalopathy or suspected seizures . Of these, 771 neonates had EEG monitoring starting at a median (IQR) age of 18.8 (8.5–39.3) hours. A total of 276 of the 771 neonates had EEG confirmed seizures, of which two neonates were not treated with ASM and 68 neonates (25.5%) had seizure termination with phenobarbital and did not require further ASM. Of the 206 neonates who had persistent seizures after phenobarbital, 5 had second line ASM protocol deviations and 49 did not have EEG during second line levetiracetam administration ( Supplementary Table S1 ). The remaining 152 neonates received levetiracetam as second line ASM under EEG monitoring and were enrolled to the study . Fig. 1 Flow chart of the study. ∗8 neonates had ongoing electrographic seizures after levetiracetam, but the clinicians decided not to administer further ASM as seizure burden was low. | 39021480_p16 | 39021480 | Results | 4.083837 | biomedical | Study | [
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The mean (SD) gestational age of these 152 neonates was 38.7 (1.6) weeks, mean (SD) birth weight was 2589 (538) grams, and 88 (57.9%) were male. Seizure etiologies were HIE in 62 (40.8%) and non-HIE in 90 (59.2%) ( Table 1 ). The non-HIE etiologies included stroke in 20, hemorrhage in 4, brain malformation in 2, sepsis in 16, metabolic in 25 (acute metabolic unresponsive to corrections 19, inborn errors of metabolism 6), and unknown causes in 23 neonates. The enrolled neonates received levetiracetam at a median (IQR) post-natal age of 32.9 (18.7–61.6) hours [range (4.1–247.5)]. Table 1 Clinical characteristics. Clinical characteristics N HIE (N = 62) N Non-HIE (N = 90) N Overall (N = 152) Gestational age, mean (SD), weeks 62 38.8 (1.4) 90 38.7 (1.8) 152 38.7 (1.6) Birth weight, mean (SD), grams 62 2621 (496) 89 2566 (567) 151 2589 (538) Male neonates, n (%) 62 35 (56.5%) 90 53 (58.9%) 152 88 (57.9%) APGAR 5 min, median (IQR) 57 6.0 (5.0–7.0) 86 8.0 (7.0–9.0) 143 7.0 (6.0–8.0) Inotropic support, n (%) 62 23 (37.1%) 76 11 (14.5%) 138 34 (24.6%) Invasive ventilation, n (%) 62 36 (58.1%) 76 18 (23.7%) 138 54 (39.1%) Death before discharge, n (%) 62 26 (41.9%) 90 17 (18.9%) 152 43 (28.2%) Seizure type a , n (%) Electrographic only seizures at all time points 60 32 (53.3%) 88 35 (39.8%) 148 67 (45.3%) Electroclinical seizures at all time points 60 5 (8.3%) 88 17 (19.3%) 148 22 (14.9%) Electrographic only seizures at one time point and electroclinical seizures at another time point. 60 23 (38.3%) 88 36 (40.9%) 148 59 (39.9%) Clinical seizure types, n (%) b Focal clonic 28 8 (28.6%) 53 29 (54.7%) 81 37 (45.7%) Tonic 28 6 (21.4%) 53 5 (9.4%) 81 11 (13.6%) Myoclonic 28 5 (17.8%) 53 6 (11.3%) 81 11 (13.6%) Spasms 28 0 (0.0%) 53 2 (3.8%) 81 2 (2.5%) Automatisms 28 10 (35.7%) 53 16 (30.2%) 81 26 (32.1%) Sequential 28 1 (3.6%) 53 1 (1.9%) 81 2 (2.5%) Autonomic 28 0 (0.0%) 53 2 (3.8%) 81 2 (2.5%) Age of starting EEG, median (IQR), hours 62 19.7 (11.7–33.9) 90 44.2 (21.7–67.6) 152 29.2 (15.9–58.6) EEG background abnormality, n (%) Normal 62 0 (0.0%) 89 2 (2.2%) 151 2 (1.3%) Mild abnormality 62 1 (1.6%) 89 22 (24.7%) 151 23 (15.2%) Moderate abnormality 62 24 (38.7%) 89 48 (53.9%) 151 72 (47.7%) Severe abnormality 62 31 (50.0%) 89 12 (13.5%) 151 43 (28.5%) Undetermined 62 6 (9.7%) 89 5 (5.6%) 151 11 (7.3%) Status epilepticus, n (%) 62 20 (32.2%) 89 27 (30.3%) 151 47 (31.1%) a Refers to seizure type after phenobarbital, once the neonate entered the study. b Data are not mutually exclusive, 1 baby may have more than 1 clinical seizure manifestation. | 39021480_p17 | 39021480 | Results | 4.17522 | biomedical | Study | [
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Among the 152 neonates enrolled, seven infants were excluded from efficacy analysis as per study criteria: six neonates with a diagnosis of inborn errors of metabolism and one infant with missing EEG data. Thus, primary outcome data were available in 145 neonates and seizure freedom occurred in 20 (13.8%; 95% CI 8.6%–20.5%) neonates following levetiracetam. Among these, 16 (80.0%) neonates responded to an initial 20 mg/kg dose, and further four (20.0%) to an additional 20 mg/kg (total 40 mg/kg). Of the 125 neonates who had persistent seizures after 40 mg/kg of levetiracetam, one infant died, one received midazolam as third line, one received phenytoin after EEG was discontinued and eight did not receive further ASM as seizure burden was considered low by the clinical team. The remaining 114 neonates received phenytoin as the third line ASM under EEG monitoring. Data on primary outcome were available in 104 out of 114 neonates as 10 neonates had early discontinuation of EEG due to clinical or logistic reasons. The primary outcome of seizure freedom occurred in 59 out of these 104 neonates (56.7%; 95% CI 46.7%–66.4%). Among these, 54 (91.5%) neonates responded to an initial 20 mg/kg dose and further five (8.5%) to an additional 20 mg/kg (total 40 mg/kg). The details of EEG monitoring of neonates analysed at ASM administration are given in Table 2 and scenarios of seizure response following administration of levetiracetam and phenytoin are shown in Supplementary Fig. S1 . Table 2 EEG characteristics of neonates analysed at antiseizure medication (ASM) administration. Characteristics N Levetiracetam (second line, N = 145) N Phenytoin (third line, N = 104) Age at ASM administration after birth (median (IQR), hours) 145 32.9 (18.7–61.6) 104 37.7 (20.5–66.0) Age at ASM administration after seizure onset (median (IQR), hours) 145 19.4 (9.2–49.6) 104 22.3 (10.4–54.6) Total duration of EEG monitoring after start of response (minutes) Median (IQR) 20 115.0 (90.0–133.7) 59 77.0 (65.0–105.0) Range 20 60–235 59 60–271 Total duration of EEG monitoring to start of next ASM in case of no response (minutes) Median (IQR) 125 88.0 (60.0–120.0) 45 120.0 (88.0–150.0) Range 125 23–435 45 30–520 Seizure type before administration of study ASM (n (%)) Electrographic only seizures at all time points 140 85 (60.7%) 99 70 (70.7%) Electroclinical seizures at all time points 140 24 (17.1%) 99 11 (11.1%) Electrographic only seizures at one time point and electroclinical seizures at another time point. 140 31 (22.1%) 99 18 (18.2%) Seizure burden before administration of study ASM (median (IQR), minutes/hour) a 145 12.0 (4.0–25.0) 104 15.0 (9.0–27.0) Status epilepticus at administration (n (%)) 145 31 (21.4%) 104 19 (18.3%) a Seizure burden calculated for the entire pretreatment period and was defined as the total duration of seizures in minutes divided by the no. of seizure hours (minutes/hour). | 39021480_p18 | 39021480 | Results | 4.144307 | biomedical | Study | [
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The median (IQR) time gap between the start of infusion of levetiracetam (20 mg/kg) and start of phenytoin was 87.0 (59.5–115.5) minutes and time gap between the start of maximal dose of levetiracetam (40 mg/kg) and phenytoin was 43.0 (30.0–59.5) minutes. The proportion of neonates who reached the efficacy endpoint (primary outcome) was greater with phenytoin as third line ASM [59/104 (56.7%; 95% CI 0.47–0.66)] compared to levetiracetam as second line ASM 20/145 [(13.8%; 95% CI 0.08–0.20)]. The attainment of seizure freedom (endpoint) with time after start of ASM is shown in Fig. 2 . Fig. 2 Seizure freedom after second line levetiracetam versus third line phenytoin. Kaplan–Meier plots showing the proportion of neonates with seizure freedom (endpoint, Y-axis) against time (minutes) from the start of initial levetiracetam 20 mg/kg infusion (X-axis) for second line levetiracetam (blue, 3A) and for neonates who also received third-line phenytoin (red, 3B). Note: The Kaplan–Meier estimates of seizure freedom are different to a simple percentage of babies with seizure freedom, as the follow-up time for babies without seizure freedom varied from 25 min to over 3 h. Neonates were censored when they reached endpoint or when the EEG monitoring stopped. | 39021480_p19 | 39021480 | Results | 4.146148 | biomedical | Study | [
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Details of seizure types and background abnormalities are provided in Table 1 . Of the 152 neonates enrolled, EEG data from one neonate was missing. Forty-seven (31.1%) of the remaining 151 neonates analysed had status epilepticus at any point during EEG monitoring. Of these, 31 (21.4%) out of 145 neonates analysed for levetiracetam response were in status epilepticus at levetiracetam administration and 19 (18.3%) out of 104 neonates analysed for phenytoin response were in status epilepticus ( Table 2 ) at phenytoin administration. Among the babies who had status epilepticus, none responded to levetiracetam (0%) whereas 2/19 (10.5%) neonates who were in status during phenytoin administration responded to phenytoin. The baseline median (IQR) seizure burden at the time of levetiracetam administration was 12.0 (4.0–25.0) minutes per hour and at the time of phenytoin administration was 15.0 (9.0–27.0) minutes per hour ( Table 2 ). The evolution of seizure burden over time and with administration of ASM is shown in Fig. 3 . Fig. 3 Seizure burden evolution over time. “ | ” = Levetiracetam, “ • ” = Phenytoin, “ X ” = age of onset of seizure (clinical or EEG), Upper panel (3A) shows the seizure burden evolution in neonates with HIE (n = 66) and lower panel (3B) shows non-HIE etiologies (n = 75). X axis- Time elapsed from the birth of baby in hours, Y axis–Each row denotes the evolution of seizure burden in a baby. Seizure burden is represented in minutes per hour and colour coded from white (0 min/h, minimum) to red (60 min/h, maximum). Blue areas represent areas where EEG monitoring was not done. The vertical black line (levetiracetam) and black dot (phenytoin) are points at which each ASM was administered, if two doses were given more than an hour apart, discrete lines are shown. | 39021480_p20 | 39021480 | Results | 4.161053 | biomedical | Study | [
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Detailed clinical seizure subclassification was performed on 148 of the 152 recruited neonates where video EEG and continuous vital sign monitoring data were available ( Table 1 ). Of these, 67 (45.3%) were electrographic-only, 22 (14.9%) were electroclinical, while 59 (39.9%) neonates had both electrographic-only and electroclinical seizures at different time points. Among the 81 (54.7%) neonates with electroclinical seizures, focal clonic seizures (45.7%) were the most common clinical manifestation followed by automatisms (32.1%). Of these 81 neonates, 58 were having electro-clinical seizures at the time of levetiracetam administration. Among these 58 neonates, 10 (17.2%) stopped seizing clinically but continued to have electrographic seizures (uncoupling) following the infusion. None of the neonates had serious adverse events like cardiac arrhythmia or sudden cardiac arrest during the time of infusion of levetiracetam or phenytoin. | 39021480_p21 | 39021480 | Results | 4.099747 | biomedical | Study | [
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Here we report, to the best of our knowledge, the largest prospective multicenter study in the world to assess electrographic response to ASM in neonatal seizures using standardized EEG acquisition and analysis protocols, and the first study from an LMIC. Levetiracetam (40 mg/kg) was associated with EEG seizure cessation within 40 min of administration in only 14% of phenobarbital unresponsive neonatal seizures. Additional treatment with phenytoin along with levetiracetam resulted in seizure freedom in further 57% of the neonates. The increase in seizure freedom may be directly related to phenytoin, delayed effects of levetiracetam or synergy. The data presented here will inform future randomised controlled trials of ASM for neonatal seizures in LMIC. | 39021480_p22 | 39021480 | Discussion | 4.117095 | biomedical | Study | [
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While there are no prospective studies evaluating efficacy of second line levetiracetam on EEG seizure freedom, efficacy data are available from few retrospective studies or subgroups of randomised controlled trials (RCT) comparing first line ASMs. In a retrospective study involving 14 neonates who had persistent seizures despite phenobarbital, Abend et al. reported that four (28%) attained complete seizure freedom. 21 In a subgroup analysis of a randomised controlled trial (Neolev-2) comparing first line phenobarbital and levetiracetam, six neonates had persistent seizures despite phenobarbital. Of these only one neonate (17%) had complete seizure freedom following levetriracetam. 16 Although the number of neonates in these studies are too small to draw any meaningful conclusions, the low efficacy of second line levetiracetam for achieving seizure freedom on EEG is consistent with the observations in our study. | 39021480_p23 | 39021480 | Discussion | 4.044739 | biomedical | Study | [
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Only two studies, both conducted over two decades ago, have reported electrographic response to phenytoin as a second line ASM. First was a landmark RCT comparing phenobarbital and phenytoin that included a subgroup of 15 neonates treated with phenytoin for non-response to phenobarbital; seizure freedom on EEG occurred in 4 (27%). 22 Another was a prospective observational study that included six neonates who had persistent seizures after phenobarbital; seizure freedom on EEG was not achieved in any of these six neonates. However, the number of neonates in these studies is too small to make meaningful comparisons about the efficacy of second line phenytoin treatment. 23 | 39021480_p24 | 39021480 | Discussion | 3.94944 | biomedical | Study | [
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In contrast, open label studies without EEG monitoring have reported much higher efficacy of levetiracetam (71%–93%) 24 , 25 in termination of clinical seizures. This may be related to observer bias inherent in open label interventions, subjectivity in the diagnosis of neonatal seizures and possibly, electroclinical uncoupling. 26 , 27 Hence pilot randomised controlled trials comparing different ASM, 13 where the investigators are neither masked to the intervention nor the outcome, are prone to serious bias. 27 In our study only 10 (17%) neonates with electroclinical seizures had uncoupling to electrographic-only seizures after administration of levetiracetam. | 39021480_p25 | 39021480 | Discussion | 4.12336 | biomedical | Study | [
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The critical importance of EEG in the evaluation of ASM efficacy is highlighted by the contrasting results of RCTs using clinical or EEG seizure freedom as primary endpoints. An open label RCT trial involving 100 neonates reported that first line levetiracetam was superior to phenobarbital in clinical seizures cessation (86% versus 62%; p < 0.01), 28 while a blinded RCT involving 83 neonates reported that phenobarbital was superior to levetiracetam (80% versus 28%; p < 0.001) in achieving seizure cessation on EEG despite the latter trial using a higher dose of levetiracetam of up to 60 mg/kg. 16 | 39021480_p26 | 39021480 | Discussion | 4.067769 | biomedical | Study | [
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It is important to note that none of the RCTs of ASM for neonatal seizures have reported neurodevelopmental outcome at 18 months or more. 29 A less effective ASM that leads to a better neurodevelopmental outcome is preferable to a highly effective ASM that adversely affects the neurodevelopment. In the original National Institute of Human Development and Child Health Neonatal Research Network hypothermia trial, 30 ASM medications were associated with adverse outcomes after HIE. 31 The confounding effects of the underlying brain injury, neonatal seizures and ASM on neurodevelopment, can be examined only in carefully designed and adequately powered double blind RCTs using EEG and robust neurodevelopmental outcome evaluation. | 39021480_p27 | 39021480 | Discussion | 4.003468 | biomedical | Study | [
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The main strength of our study is the large number of neonates we were able to enrol with continuous video EEG monitoring, particularly in a LMIC setting. We also had trained neonatal neurology fellows and technicians to allow real time EEG reporting and feedback to the clinical team. We carefully annotated the ASM start points on the EEG which enabled us to accurately quantify measures such as seizure burden before and after the administration of the ASM. Building on this work, we have established a Collaborative Neonatal Neuroprotection Trial platform in South Asia (CONNECTIONS) to conduct large multi-country trials of ASM and other neuroprotective therapies. | 39021480_p28 | 39021480 | Discussion | 3.886719 | biomedical | Study | [
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Our study had several limitations. Firstly, our study design was observational and direct comparisons cannot be made as additional confounders and temporal changes may have influenced the efficacy of ASM unequally. Thus, neonates in levetiracetam group had both phenobarbital and levetiracetam, while those in the phenytoin group had phenobarbital, levetiracetam and phenytoin. Therefore, synergy or later effect of levetiracetam could have amplified the efficacy of phenytoin. Nevertheless, poor (14%) seizure cessation even after 40 min of levetiracetam administration is a concern, particularly as 31% of the neonates were in status epilepticus in our study. Furthermore, neonatal seizures tend to be more refractory to treatment over time. 32 Despite this phenytoin as a third line was associated with more seizure freedom than second line levetiracetam. Although seizures in HIE tend to peak around 24 h before naturally decreasing by 72 h, the median time interval between full dose of levetiracetam and phenytoin was too short (43 min) for these temporal changes to modify the treatment efficacy in our study. | 39021480_p29 | 39021480 | Discussion | 4.142481 | biomedical | Study | [
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Secondly, although our study protocol required the levetiracetam infusion to be completed within 30 min, we did not collect the exact time when the infusion was completed. To account for any potential delays, we used seizure freedom within 40 min from the start of the infusion. | 39021480_p30 | 39021480 | Discussion | 2.880213 | biomedical | Study | [
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Third, we used a maximal dose of 40 mg/kg of levetiracetam as safety data on high dose levetiracetam are lacking, and hence cannot exclude a better efficacy with higher doses. 33 | 39021480_p31 | 39021480 | Discussion | 2.794584 | biomedical | Study | [
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Fourth, our primary outcome was based on seizure freedom within 40 min of the administration and not 24 h as in earlier studies from high-income countries. 16 Temporary cessation of seizures is frequent after ASM is given. 34 Hence in clinical trials, EEG monitoring is recommended to continue for at least 24 h after seizure cessation to detect any possible recrudescence of seizures, and because seizures frequently wax and wane over hours. However, acquiring continuous EEG monitoring over 24 h was logistically challenging as electrical and movement artefacts were common in Indian neonatal units. Hence EEG technicians or neonatal fellows trained in neonatal EEG had to be present at the bedside to ensure data quality in our study. | 39021480_p32 | 39021480 | Discussion | 4.067497 | biomedical | Study | [
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Finally, whole body-hypothermia was being offered at the participating sites before the publication a randomised controlled trial (Hypothermia for Encephalopathy in Low and Middle-Income countries; HELIX) 35 reporting lack of neuroprotection and increased mortality with this treatment, and hence was de-implemented. Thus, only one neonate received whole-body hypothermia in this study and the body temperature of other neonates was maintained in the normothermic range. Whole-body hypothermia may reduce seizure burden, and lower the renal clearance of levetiracetam, 36 , 37 all of which may modify the treatment response. The disease severity among neonates recruited to our study was also high compared to high-income countries, as observed by the high rates of status epilepticus and mortality before discharge. Therefore, the results may not be generalisable to neonates in high-income countries or those treated with whole-body hypothermia. | 39021480_p33 | 39021480 | Discussion | 4.084385 | biomedical | Study | [
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In this large multicenter observational study involving neonates with seizures unresponsive to phenobarbital, levetiracetam at a maximal dose of 40 mg/kg, was associated with attainment of seizure freedom on EEG only in 14% of the neonates by 40 min after the start of the infusion. As 31% of the neonates were in status epilepticus, low efficacy of levetiracetam in EEG seizure cessation is of concern. Additional treatment with phenytoin along with levetiracetam resulted in seizure freedom in further 57% of the neonates. The increase in seizure freedom may be directly related to phenytoin, delayed effects of levetiracetam or synergy. | 39021480_p34 | 39021480 | Discussion | 4.145103 | biomedical | Study | [
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VK recruited babies, interpreted the EEG data and wrote the first draft under supervision of RP and ST. VK, VU, HV, MKA, and PW recruited babies, interpreted the EEG data. SF, SC, SD, MK, VKG, and AVT supervised the site recruitments. PMo, CB, RG, PM and SP assisted in trial management and preparation of the manuscript. CRN, JHC, SS assisted in protocol development, interpretation of the data and preparation of the manuscript. PB was responsible for all statistical analysis. RP assisted in protocol development, reviewed all EEGs and was responsible for the interpretation and analysis. All authors approved the final version of the manuscript. ST obtained funding, supervised all aspects of the study including data analysis and interpretation, preparation of the manuscript and had final responsibility for the decision to submit for publication. | 39021480_p35 | 39021480 | Contributors | 0.896363 | other | Other | [
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Anonymised participant data used in this study will be available from the corresponding author after approval of a proposal with a signed data access agreement. | 39021480_p36 | 39021480 | Data sharing statement | 1.00546 | biomedical | Other | [
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Helen Cross has received institutional renumeration from Zogenix, Union Chimique Belge (UCB), Marinius, Stroke Therapeutics, Ultragenyx, GW Pharma, Jazz, Biocodex for educational symposium and advisory board activities and renumeration for administrative support from International League Against Epilepsy at the President. Ronit Pressler has received institutional funding from UCB and personal renumeration from Kephala and Natus for lectures. The authors declare no conflict of interest. | 39021480_p37 | 39021480 | Declaration of interests | 0.997558 | other | Other | [
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The capacity to heal wounds is essential for organisms to endure and thrive despite an occasionally hostile environment. Organisms throughout the animal kingdom can heal wounds, but mammalian wound healing has been studied most intensively because of its medical relevance. Wound healing must occur to restore health after trauma or surgery, or in conditions such as cancer or peptic ulcers in which internal processes cause tissue damage. Mammalian epithelial tissues display a characteristic set of responses to tissue damage, including the rapid formation of a blood clot at the site of injury, followed by spreading of the damaged epithelium across the wound gap to restore tissue integrity . However, there are significant differences in the wound healing response depending on the specific tissue affected, its developmental stage, and the nature of the damage. For example, damaged fetal epidermis heals without leaving a scar , and a few adult tissues, including human liver, can regenerate large portions of the damaged tissue . Some wounds, such as the common foot ulcers of diabetics, heal slowly or not at all , whereas others display an exaggerated response that results in disfiguring keloid scars . One important goal of wound healing research is to find ways to speed or alter the healing process. Another is to understand the fundamental cellular and molecular mechanisms by which cells sense tissue damage and signal to neighboring healthy cells to contain and repair it. | 15269788_p0 | 15269788 | Introduction | 4.376995 | biomedical | Review | [
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Cellular studies of mammalian wound healing have shown that it is a complex process that takes weeks to complete and involves not just the damaged epithelial cells and their neighbors, but also fibroblasts and blood vessels in the underlying stroma, and inflammatory cells that are recruited to the wound site . Only the first step in mammalian wound healing, the proteolytic cascade that culminates in fibrin deposition and clot formation, is well understood at the molecular level . As the clot forms, platelets bound to it and to the damaged tissue release additional procoagulant proteins as well as growth factors and chemokines that can attract neutrophils and monocytes that mediate an early inflammatory response. Keratinocytes at the wound margin become activated, break down their cell junctions, and assume a lamellipodial crawling morphology as they spread across the wound site to restore epithelial integrity . The early inflammatory cells release additional signals that can attract and activate fibroblasts, macrophages, and blood vessel endothelial cells. These cells infiltrate the wound site and form a specialized stroma called granulation tissue, which facilitates reepithelialization, helps contract the wound, and is later remodeled to form the scar. | 15269788_p1 | 15269788 | Introduction | 4.778214 | biomedical | Study | [
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Although many different cell types are present at the wound site, and dozens of signaling molecules, receptors, matrix proteins, and proteases are known to be expressed during the healing process , their roles in the process have been difficult to establish. This difficulty is due to the cellular and molecular complexity of wound healing and the challenges in manipulating wound gene expression and function in vivo. Hence, models of gene function in wound healing derive primarily from results of gene expression studies at wound sites, application of exogenous gene products to wounds, and studies in simple cell culture models such as keratinocyte monolayers. Analyses of wound healing defects in mouse knockouts of candidate genes have also begun to provide insight into the genes' roles in the process . However, some of the genetic results challenge fundamental aspects of the prevailing models . | 15269788_p2 | 15269788 | Introduction | 4.15592 | biomedical | Study | [
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The establishment of simpler, more tractable genetic systems to study wound healing could allow systematic genetic dissection of the process in vivo and complement studies in vertebrates and clinical settings. Over a half century ago, Wigglesworth demonstrated that the large hemipteran insect Rhodnius prolixus has a robust wound healing response . He characterized the response by light microscopy and described the proliferation and spreading of epidermal cells and the accumulation of blood cells (hemocytes) at the wound site. Since this pioneering work, only a few follow-up studies have appeared . There has been little work on other insects aside from a number of studies of wound healing during imaginal disc and leg regeneration and the recent discoveries that Drosophila embryos undergo a scarless wound healing process involving actin cable formation and filopodial extension and that wounded adult cells activate the Jun N-terminal kinase (JNK) signaling pathway . Some attention has also focused on melanization, the formation of a heteropolymer of orthoquinones generated by phenoloxidase-catalyzed oxidation of mono- and diphenols that accompanies certain infections, tumors, and wound healing . | 15269788_p3 | 15269788 | Introduction | 4.356037 | biomedical | Review | [
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We set out to investigate wound healing in Drosophila melanogaster because of the powerful genetic and genomic approaches available in this organism. These approaches have elucidated the molecular pathways that control many developmental and physiological processes. For example, genetic studies revealed a prominent role for a JNK signaling pathway in Drosophila dorsal closure, a developmentally programmed spreading of the embryonic epidermis . This process resembles epithelial spreading during vertebrate wound healing, and indeed this similarity and the expression patterns of JNK pathway transcription factors near wounds prompted two recent genetic studies of JNK pathway activity in adult wound healing . | 15269788_p4 | 15269788 | Introduction | 4.198171 | biomedical | Study | [
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In this paper, we describe the cellular events and genetic requirements of epidermal wound healing in Drosophila larvae. A simple puncture wound assay was developed, and we use it to show that a plug rapidly forms at the wound site and subsequently melanizes to form a scab. We describe how epidermal cells surrounding the plug orient toward it and fuse to form a syncytium, and how the cells spread along and through the plug to reestablish epithelial continuity. We then use JNK pathway reporters and genetic analysis to demonstrate the induction and function of the JNK pathway in the process, and we use mutants that block scab formation, and a scabless wounding procedure, to elucidate the function of the scab. The results demonstrate that the cellular responses of wound healing are under separate genetic control, and that they are coordinated by multiple signals emanating from the wound site, including a negative feedback signal between scab formation and the JNK pathway. This establishes a tractable genetic system to study postembryonic wound healing, and the cellular and molecular parallels with vertebrate wound healing suggest that some of the fundamental steps in the process are evolutionarily conserved. | 15269788_p5 | 15269788 | Introduction | 4.455226 | biomedical | Study | [
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A puncture wounding procedure was developed in which early third instar (L3) Drosophila larvae were lightly anesthetized and then stabbed with a 0.1-mm–diameter steel needle, about the size of six epidermal cells . To ensure reproducibility, larvae were always stabbed at the dorsal midline halfway between the hair stripes of abdominal segments A3 or A4. Wounding did not cause a developmental arrest, because the wounded larvae continued to grow and pupariated 48 h after wounding, similar to mock-wounded controls , and 90% or more of the wounded larvae survived the procedure (see below). We then analyzed the major morphological, cellular, and molecular events of healing by visualizing wounds at different stages of healing in live and heat-killed whole-mount larvae, in histochemically or immunostained larval fillets, and in sections through wounds that we examined by transmission electron microscopy (TEM) . | 15269788_p6 | 15269788 | A Larval Epidermal Wound Healing Assay | 4.164235 | biomedical | Study | [
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Unwounded larvae have a semitransparent white cuticular surface with rare or no blemishes . Beneath the cuticle is the epidermis , an epithelial monolayer that secretes the cuticle at its apical (external) surface and is lined by a basal lamina along its basal surface . Immediately after puncture wounding, a variable amount of blood (hemolymph) escapes from the wound site (data not shown). Within 10–15 min, the wound site begins to darken and a plug forms in the gap . The plug is composed of debris, presumably the remnants of necrotic cells damaged by wounding that are disorganized and highly vesiculated and not bound by a cell membrane or basal lamina . The plug may also contain blood coagulation products (see Discussion). | 15269788_p7 | 15269788 | Bleeding and Scab Formation at the Wound Site | 4.218618 | biomedical | Study | [
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Over the next 24 h, the outer part of the plug is converted into a scab. This part of the plug becomes electron dense as the scab enlarges and darkens , presumably due to a melanization reaction. Melanization affects all of the external structures at the wound site including the debris, the edges of the damaged cuticle , and even entrapped tissues such as tracheae . | 15269788_p8 | 15269788 | Bleeding and Scab Formation at the Wound Site | 3.753737 | biomedical | Other | [
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By 2 or 3 d after wounding, debris is cleared, the scab resolves, and the exterior of the animal resumes a nearly normal appearance . Epidermal cells that grow back across the wound gap (see below) appear to participate in debris clearance, because they extend processes that engulf the debris and contain within their cytoplasm vesiculated material resembling debris . Other components of the plug and scab may be degraded extracellularly or passively shed from the wound site. | 15269788_p9 | 15269788 | Bleeding and Scab Formation at the Wound Site | 4.171745 | biomedical | Study | [
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The response of epidermal cells to wounding was examined in transgenic larvae in which epidermal cell nuclei were labeled with green fluorescent protein (GFP) and cell membranes were immunostained for the basolateral membrane marker Fasciclin III or the septate junction protein Coracle . Epidermal cells at the wound site underwent two dramatic morphological changes in the several hours following wounding. First, beginning about a half hour after wounding, cells at the wound margin began to elongate and orient toward the wound, often tapering toward the wound site . Second, these cells fused with each other to form a syncytium. Normally, epidermal cells are mononuclear . However, as early as 1 h after wounding, the radially oriented plasma membrane domains (parallel to the long cell axis) began to break down as the circumferential domains joined, creating multinucleate cells around the wound. This can be seen in the Fasciclin III and Coracle stains, which showed incomplete or absent radial domains of plasma membrane staining; the loss of these membrane domains was sometimes accompanied by scattered puncta of staining in the cytoplasm, which may be membrane breakdown intermediates . TEM analysis confirmed the absence of plasma membrane between epidermal nuclei beneath and adjacent to the wound site . Syncytia were nearly always present by 4 h after wounding. | 15269788_p10 | 15269788 | Epidermal Cells Orient toward the Wound and Fuse to Form a Syncytium | 4.44887 | biomedical | Study | [
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As healing progressed, the polarization and fusion of epidermal cells spread outward from the wound. As cells bordering the wound fused, the more-peripheral cells just beyond the syncytium began to polarize toward the wound . Some of these cells apparently proceed to fuse with the central syncytium, because the average number of nuclei per syncytium increased over the 2 d following wounding, creating a large syncytium with as many as 30 nuclei surrounding the wound . Epidermal cell or nuclear division do not contribute to growth of the syncytium, because neither was detected around the wound site or elsewhere in the epidermis by immunostaining for phosphorylated histone H3, a marker of condensed mitotic chromosomes, 4–24 h after wounding (data not shown). Highly asymmetrical syncytia like the one shown in Figure 3 D probably represent cases in which a subset of polarized peripheral cells had fused with the central syncytium. Peripheral cells may fuse to each other before fusing to the central syncytium, because satellite syncytia separate from the central syncytium were occasionally observed. | 15269788_p11 | 15269788 | Epidermal Cells Orient toward the Wound and Fuse to Form a Syncytium | 4.259939 | biomedical | Study | [
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A key step in wound healing is the closure of the epidermal gap and reestablishment of epithelial integrity. By 2 h after wounding, the epidermis was still discontinuous but the breach was filled by the plug and developing scab . Ultrastructural studies showed that during the next 6 h, as the epidermal cells oriented toward the puncture site and fused to form a syncytium, they also spread along and through the plug, led by lamellipodial extensions , until epithelial continuity was reestablished . No multicellular actin cable indicative of the “purse string” closure mechanism of embryonic wound healing was observed in the spreading cells (see Materials and Methods ). A thin basal lamina was present along the length of the lamellipodia , suggesting that basal lamina is synthesized by the cells before or during their migration. Following reepithelialization, new cuticle was secreted . By 24 h after wounding, a thick new cuticle layer was present that was continuous with the old cuticle at the wound margin . Most of the wound plug debris ended up outside the new cuticle layer and eventually melanized to form scab , although occasionally some was left beneath the epidermis and was later phagocytosed or degraded . | 15269788_p12 | 15269788 | Epidermal Cells Spread along and through the Plug to Reestablish Epithelial Integrity | 4.514936 | biomedical | Study | [
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To elucidate the genetic control and interdependence of the cellular events of wound healing, we investigated the activity and function of the JNK signaling pathway in the process . The epidermal spreading in some ways resembles the epidermal spreading of dorsal closure, which depends on the JNK pathway. During dorsal closure, the mitogen-activated protein kinase kinase kinase kinase Misshapen is activated, triggering a phosphorylation cascade that ultimately activates the JNK Basket . Basket phosphorylates the Drosophila Jun and Drosophila Fos transcription factors , thus inducing expression of puckered (puc), which encodes a phosphatase that negatively regulates Basket, and other targets . To test for JNK pathway activation in the larval puncture wound assay, we assayed expression of lacZ transcriptional reporters of puc and misshapen (msn), two genes induced by JNK pathway activation in other contexts . | 15269788_p13 | 15269788 | The JNK Pathway Is Activated in a Gradient and Promotes Reepithelialization | 4.319146 | biomedical | Study | [
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In unwounded larval epidermis, there was little or no detectable expression of either the msn or the puc reporter . However, within 1 h after wounding, expression of both reporters was readily detected in epidermal cells surrounding the wound, and by 4 h both exhibited robust expression . The msn and puc reporters were induced in large, roughly symmetrical zones extending three to seven cell diameters out from the puncture site. Within each zone, the reporters were expressed in a gradient, with cells closest to the puncture site exhibiting the highest level of expression, suggesting that the reporters are induced by a signal emanating from the wound site. The zone of expression of the msn reporter was typically broader than that of puc, perhaps because it is more sensitive to the inducing signal. Expression of both reporters peaked between 4 and 8 h after wounding and declined thereafter, with expression restricting to cells closest to the wound . | 15269788_p14 | 15269788 | The JNK Pathway Is Activated in a Gradient and Promotes Reepithelialization | 4.276046 | biomedical | Study | [
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To determine the function of JNK pathway induction, we analyzed wound healing in larvae in which the JNK pathway was inactivated. Because null mutations in JNK pathway genes block dorsal closure and are embryonic lethal, we selectively inhibited the pathway in larval epidermis by expressing a dominant-negative form of Basket JNK (upstream activation sequence-basket dominant negative [UAS-bsk DN ]) under the control of the A58-Gal4 driver, an epidermal-specific driver that turns on early in larval development. UAS-bsk DN was used because it is the most potent JNK pathway inhibitor available (see Materials and Methods ): it gave a severe dorsal closure phenotype and lethality when expressed in the embryonic epidermis using e22c-Gal4 or 69B-Gal4 drivers. By contrast, larvae expressing UAS-bsk DN under control of the A58-Gal4 driver were viable and active and did not display any morphological abnormalities, suggesting that the JNK pathway does not play a critical role in the larval epidermis under normal environmental conditions. However, following wounding, induction of the msn reporter was almost completely abolished , and the wound healing process was dramatically affected. | 15269788_p15 | 15269788 | The JNK Pathway Is Activated in a Gradient and Promotes Reepithelialization | 4.269151 | biomedical | Study | [
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We analyzed the effect of JNK pathway inhibition on wound healing using the assays used for wild-type larvae. There were no detectable defects in the early steps in wound healing, including scab formation, epidermal cell orientation toward the wound, and epidermal cell fusion to form a syncytium . However, ultrastructural analysis showed that reepithelialization was blocked or defective, with no cytoplasmic processes or only extremely fine or distorted processes and no new cuticle synthesis beneath the scab 16 h after wounding . | 15269788_p16 | 15269788 | The JNK Pathway Is Activated in a Gradient and Promotes Reepithelialization | 4.19495 | biomedical | Study | [
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To further test the requirement of the JNK pathway in reepithelialization, we analyzed larvae in which a portion of the epidermis was abraded by a nonpenetrating pinch wounding procedure (described further below) that leaves a much larger gap in the epidermis than does a fine puncture wound and hence provides a more rigorous test of wound reepithelialization . In control larvae in which the JNK pathway was not inhibited, the epidermis spread to close the gap, and full reepithelialization was evident within 24 h after wounding . By contrast, in larvae in which the JNK pathway was inhibited, the epidermis did not spread, and a large gap remained . We conclude that induction of the JNK pathway promotes spreading and reepithelialization of the larval epidermis but appears to be dispensable for other steps in wound healing, including scab formation, cell orientation, and cell fusion. | 15269788_p17 | 15269788 | The JNK Pathway Is Activated in a Gradient and Promotes Reepithelialization | 4.254981 | biomedical | Study | [
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To investigate the role of the scab in puncture wound healing, we sought ways to block scab formation genetically . Crystal cells are a special type of blood cell that contain distinctive, crystal-like intracellular inclusions and have long been hypothesized to play a role in melanization responses such as those in scab formation . The gene lozenge (lz) encodes a transcription factor required for development of the crystal cell lineage , and crystal cells are severely reduced or absent in lz r15 homozygous or hemizygous larvae . The lz r15 mutant larvae failed to form a scab detectable by light microscopy , and TEM analysis showed a diffuse plug at the wound site instead of the consolidated, electron-dense plug and scab that are normally present 24 h after wounding . This defect in scab formation is likely due to the effect of lz r15 on crystal cells, and not some other effect of the mutation, because scab formation was also inhibited in larvae homozygous for Black cells (Bc) (data not shown), a mutation that alters crystal cell morphology and eliminates serum phenoloxidase activity . We conclude that crystal cells are required to consolidate and melanize the plug to form a scab during wound healing. | 15269788_p18 | 15269788 | Crystal Cells Promote Scab Formation | 4.364606 | biomedical | Study | [
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Untreated lz r15 larvae were viable and active, but few survived the normal puncture wound procedure . By 4 h after wounding, only 55% of lz r15 larvae were alive, and by 24 h only 15% survived, most of which were sluggish and flaccid. By contrast, 85% or more of the lz + control larvae survived the wounding procedure. Thus, scab formation is critical for healing puncture wounds. | 15269788_p19 | 15269788 | Crystal Cells Promote Scab Formation | 4.004946 | biomedical | Study | [
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We next investigated the cellular events of wound healing in lz r15 larvae, using the methods described above for wild-type and JNK pathway mutants, except that sharper pins were used for wounding to increase survival and allow analysis of the later stages of wound healing. Most of the cellular responses to wounding appeared to initiate in lz r15 mutants, although they did not progress normally. Epidermal cell fusion occurred, but the syncytium often occupied a greater area than in control larvae . The surrounding epidermal cells also appeared to organize around the wound, but their cell borders were slack and wavy, even several cell diameters out from the wound, making it difficult to assess whether they had oriented toward the wound . A similar though less severe “wavy border” phenotype was observed in Bc mutant larvae. TEM analysis revealed that the epidermal cells up to 200 μm or more beyond the wound margin separated from the overlying cuticle around the wound . However, the detached cells extended numerous fine cellular processes in an apparent attempt to close the wound. Sometimes the edges of the punctured epidermis met to restore epithelial integrity, but in most cases they did not . | 15269788_p20 | 15269788 | The Scab Stabilizes the Wound Site and Prevents Superinduction of the JNK Pathway | 4.264483 | biomedical | Study | [
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The lz r15 mutation also caused superinduction of the JNK pathway reporters. Although the basal expression level of the msn and puc reporters in unwounded epidermis was unchanged, both were expressed at higher levels and in an expanded zone around the wound site at 3, 6, and 24 h after wounding . A similar effect was observed in Bc mutants. Thus, scab formation limits induction of the JNK pathway around puncture wounds. | 15269788_p21 | 15269788 | The Scab Stabilizes the Wound Site and Prevents Superinduction of the JNK Pathway | 4.18782 | biomedical | Study | [
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To further investigate the role of the scab in wound healing, a scabless wound healing procedure was developed. The larval cuticle was gently pinched with dissecting forceps, leaving the cuticle intact but abrading a patch of epidermal cells from its inner surface . Although these pinch wounds did not bleed or form scabs, the epidermal cells at the wound site underwent many of the same responses seen at puncture wounds. Many cells at the wound margin oriented toward the wound, and some fused with neighboring cells to form syncytia . Also, the msn reporter was induced in a gradient in the cells surrounding the wound , and the cells spread to close the wound gap within 24 h . Thus, each of the major epidermal cell responses to wounding can occur normally in the absence of a scab, provided the cuticle remains intact. Indeed, the primary function of the scab may be to restore integrity to the cuticle and wound site, because lz r15 mutant larvae did not display any defects in the healing of pinch wounds: epidermal cells around the wound polarized and fused like in lz + controls, the JNK pathway reporters were induced at their normal levels and in their normal domain around the wound site, and the epidermal cells spread across the wound and healed with normal kinetics . Thus, the critical function of the scab appears to be to provide stability to the damaged cuticle and wound site, and the defects observed in the epidermal cell responses following puncture wounding of lz r15 mutants most likely arise secondarily to the persistent instability of the wound site. | 15269788_p22 | 15269788 | The Scab Stabilizes the Wound Site and Prevents Superinduction of the JNK Pathway | 4.41698 | biomedical | Study | [
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We established an epidermal wound healing assay in Drosophila larvae and elucidated the cellular events and genetic requirements of the healing process. Following puncture wounding, the damaged epidermal cells and their neighbors execute a series of responses that limit blood loss and restore integrity to the epidermis and overlying cuticle . Shortly after wounding, a plug forms in the wound gap. Over the next several hours, the outer portion of the plug melanizes to form a scab, and epidermal cells at the wound margin begin to elongate and orient toward the wound. They then fuse with each other to form a syncytium surrounding the wound. Subsequently, more-peripheral cells orient toward and fuse with the central syncytium. No proliferation of epidermal cells or actin cable formation was detected at the wound site. Instead, the epidermal cells surrounding the wound migrate along or through the plug to restore continuity of the epithelium and its basal lamina and cuticle lining. | 15269788_p23 | 15269788 | Discussion | 4.336199 | biomedical | Study | [
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Each of these responses—scab formation, epidermal cell orientation and fusion, and epidermal spreading and reepithelialization—occurs at characteristic times and positions during wound healing. However, our results suggest that these responses are under separate genetic control and are not contingently coupled . Scab formation is dependent on crystal cells and is inhibited by the lz r15 and Bc mutations. Epidermal spreading and reepithelialization require bsk and JNK pathway activity, which is rapidly induced in epidermal cells surrounding the wound site. Epidermal cell orientation and fusion can proceed even in the absence of scab formation or JNK pathway activity. Although the different responses have distinct genetic requirements and can initiate independently of each other, we identified one important interaction between them. In lz r15 and Bc mutants, reepithelialization initiated but was not always completed, and the JNK pathway was hyperinduced, implying that the scab normally facilitates reepithelialization and restrains JNK activation. | 15269788_p24 | 15269788 | Discussion | 4.44784 | biomedical | Study | [
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] | en | 0.999998 |
Below, we discuss the mechanisms and functions of each of these wound healing responses and the signals that trigger them, and suggest a mechanistic basis for the observed interaction between scab formation, reepithelialization, and JNK activation. We also compare wound healing in Drosophila with the related processes in mammals and speculate on their evolutionary relationship. | 15269788_p25 | 15269788 | Discussion | 3.989797 | biomedical | Review | [
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The wound plug that forms shortly after puncture wounding contains cell debris, and it may also contain blood coagulation products like those identified in other arthropods and recently in Drosophila . Over the next few hours the plug rapidly darkens and becomes electron dense, presumably the result of a melanization reaction. Although the nature and extent of melanin cross-linking to tissues has not been studied, it seems likely that the polymer links to wound plug components and cuticle to strengthen and stabilize the wound site. | 15269788_p26 | 15269788 | Formation and Function of the Scab | 4.191463 | biomedical | Study | [
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] | en | 0.999998 |
Subsets and Splits