Molecular stratification of the human fetal vaginal epithelium by spatial transcriptome analysis.

The human vaginal epithelium is a crucial component of numerous reproductive processes and serves as a vital protective barrier against pathogenic invasion. Despite its significance, a comprehensive exploration of its molecular profiles, including molecular expression and distribution across its multiple layers, has not been performed. In this study, we perform a spatial transcriptomic analysis within the vaginal wall of human fetuses to fill this knowledge gap. We successfully categorize the vaginal epithelium into four distinct zones based on transcriptomic profiles and anatomical features. This approach reveals unique transcriptomic signatures within these regions, allowing us to identify differentially expressed genes and uncover novel markers for distinct regions of the vaginal epithelium. Additionally, our findings highlight the varied expressions of keratin ( KRT) genes across different zones of the vaginal epithelium, with a gradual shift in expression patterns observed from the basal layer to the surface/superficial layer. This suggests a potential differentiation trajectory of the human vaginal epithelium, shedding light on the dynamic nature of this tissue. Furthermore, abundant biological processes are found to be enriched in the basal zone by KEGG pathway analysis, indicating an active state of the basal zone cells. Subsequently, the expressions of latent stem cell markers in the basal zone are identified. In summary, our research provides a crucial understanding of human vaginal epithelial cells and the complex mechanisms of the vaginal mucosa, with potential applications in vaginal reconstruction and drug delivery, making this atlas a valuable tool for future research in women's health and reproductive medicine.

Decreased serum soluble programmed cell death ligand-1 level as a potential biomarker for missed miscarriage.

STUDY QUESTION: Can maternal serum levels of soluble programmed cell death-1 (sPD-1) and its ligand (sPD-L1) serve as biomarkers for missed miscarriage (MM)? SUMMARY ANSWER: Serum sPD-L1 levels are significantly decreased in MM patients and may serve as a potential predictive biomarker for miscarriage. WHAT IS KNOWN ALREADY: Programmed cell death-1 (PD-1) and its ligand (PD-L1) comprise important immune inhibitory checkpoint signaling to maintain pregnancy. Their soluble forms are detectable in human circulation and are associated with immunosuppression. STUDY DESIGN, SIZE, DURATION: Three independent cohorts attending tertiary referral hospitals were studied. The first (discovery) cohort was cross-sectional and included MM patients and healthy pregnant (HP) women matched on BMI. The second validation cohort contained MM patients and women with legally induced abortion (IA). The third prospective observational study recruited subjects requiring IVF treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: In the discovery cohort, we enrolled 108 MM patients and 115 HP women who had a full-term pregnancy at 6-14 weeks of gestation. In the validation cohort, we recruited 25 MM patients and 25 women with IA. Blood samples were collected at the first prenatal visit for HP women or on the day of dilatation and curettage surgery (D&C) for MM and IA subjects to determine serum sPD-1 and sPD-L1 levels. Placenta samples were harvested during the D&C within the validation cohort to measure gene and protein expression. The prospective cohort collected serial blood samples weekly from 75 volunteers with embryo transfer (ET) after IVF. MAIN RESULTS AND THE ROLE OF CHANCE: Circulating sPD-L1 levels were reduced by 50% in patients with MM (55.7 ± 16.04 pg/ml) compared to HP controls (106.7 ± 58.46 pg/ml, P < 0.001) and the difference remained significant after adjusting for maternal age and gestational age, whereas no significant differences in sPD-1 level were observed. Likewise, serum sPD-L1 was lower in MM patients than in IA subjects and accompanied by downregulated PD-L1-related gene expression levels in the placenta. In the IVF cohort, applying the changing rate of sPD-L1 level after ET achieved a predictive performance for miscarriage with receiver operating characteristics = 0.73 (95% CI: 0.57-0.88, P < 0.01). LIMITATIONS, REASONS FOR CAUTION: The study was mainly confined to East Asian pregnant women. Further large prospective pregnancy cohorts are required to validate the predictive performance of sPD-L1 on miscarriage. WIDER IMPLICATIONS OF THE FINDINGS: Reduced circulating sPD-L1 level and downregulated placental PD-L1 expression in miscarriage indicate that dysfunction in PD-L1 signals is a potential underlying mechanism for pregnancy loss. Our findings further extend the importance of the PD-L1 axis in pregnancy maintenance in early pregnancy. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by grants from the Subject Innovation Team of Shaanxi University of Chinese Medicine (2019-Y502), General Research Fund (14122021), and Key Laboratory of Model Animal Phenotyping and Basic Research in Metabolic Diseases (2018KSYS003). The authors declare that they have no competing interests to be disclosed. TRIAL REGISTRATION NUMBER: N/A.

Enzymatic preparation and potential applications of agar oligosaccharides: a review.

Oligosaccharides derived from agar, that is, agarooligosaccharides and neoagarooligosaccharides, have demonstrated various kinds of bioactivities which have been utilized in a variety of fields. Enzymatic hydrolysis is a feasible approach that principally allows for obtaining specific agar oligosaccharides in a sustainable way at an industrial scale. This review summarizes recent technologies employed to improve the properties of agarase. Additionally, the relationship between the degree of polymerization, bioactivities, and potential applications of agar-derived oligosaccharides for pharmaceutical, food, cosmetic, and agricultural industries are discussed. Engineered agarase exhibited general improvement of enzymatic performance, which is mostly achieved by truncation. Rational and semi-rational design assisted by computational methods present the latest strategy for agarase improvement with greatest potential to satisfy future industrial needs. Agarase immobilized on magnetic Fe3O4 nanoparticles via covalent bond formation showed characteristics well suited for industry. Additionally, albeit with the relationship between the degree of polymerization and versatile bioactivities like anti-oxidants, anti-inflammatory, anti-microbial agents, prebiotics and in skin care of agar-derived oligosaccharides are discussed here, further researches are still needed to unravel the complicated relationship between bioactivity and structure of the different oligosaccharides.

Heterologous Expression of Phycocyanobilin in Escherichia coli and Determination of Its Antioxidant Capacity In Vitro.

Phycocyanobilin (PCB) is a blue pigment with antioxidant, anti-inflammatory, and anticancer properties. It is used in the medical and cosmetic industries. In this study, a high-expression plasmid, pET-30a-PCB, was constructed for expression of PCB in Escherichia coli BL21(DE3). The PCB was analyzed using UV-visible absorption spectrum, MALDI-TOF-MS, and fluorescence spectra. The stability and half-life of PCB in different serum were determined. The yield of PCB was optimized through single-factor and orthogonal experiments. The optimal expression conditions were determined as a lactose concentration of 5 mmol/L, an induction time of 8 h, an induction temperature of 27 °C, and an induction duration of 22 h. PCB yield of 6.5 mg/L was achieved and subsequently purified using nickel-affinity chromatography. The purified PCB was quantified indirectly using Hist-tag ELISA detection, and the concentration was 11.66 µg/L. In the range of 0-33 µg/mL, the total antioxidant capacity and reducing the capacity of PCB were stronger than Vitamin E (Ve), with 1,1-diphenyl-2-picrylhydrazil (DPPH) scavenging reaching up to 87.07%, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) free radical (ABTS) scavenging up to 100%, hydroxyl radicals (·OH) scavenging up to 64.19%, hydrogen peroxide (H2O2) scavenging up to 78.75%, This study provides theoretical evidence for PCB as a potent antioxidant.

Vaginal reconstruction by collagen scaffolds loaded with vaginal epithelial and smooth muscle cells in pigs.

In women, a healthy and functional vagina is important for the maintenance of a good quality of life. Various factors, including congenital anomalies, cancer, trauma, infections, inflammation, or iatrogenic injuries, can lead to damage or loss of the vaginal structure, necessitating repair or replacement. Often, such reconstruction procedures involve the use of nonvaginal tissue substitutes, like segments of the large intestine or skin, which are less than ideal both anatomically and functionally. Therefore, there is an urgent need to develop new methods of vaginal reconstruction. In this study, we established a new method for isolation and expansion of vaginal epithelial and smooth muscle cells. Subsequently, collagen scaffolds designed for vaginal reconstruction were loaded with vaginal epithelial and smooth muscle cells in vitro and tested in vivo using the vaginal excision pig model. The results showed that the collagen scaffold loaded with vaginal epithelial and smooth muscle cells significantly promotes the reconstruction of the vagina compared with small intestinal submucosa (SIS) membrane or bare collagen scaffold. Notably, the reconstructed vaginal tissues exhibit remarkable similarity to their normal counterparts, encompassing not only the vaginal epithelium and smooth muscle but also the intricate networks of blood vessels and nerves. These compelling results underscore the feasibility of a tissue engineering approach in vaginal reconstruction, offering promising prospects for improving the quality of life in affected individuals.

Traditional Chinese medicine for insomnia: Recommendation mapping of the global clinical guidelines.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese Medicine (TCM) represents a rich repository of empirically-developed traditional medicines. The findings call for more rigorous study into the efficacy, safety, and mechanisms of action of TCM remedies to strengthen the evidence base. AIM OF THE STUDY: To systematically review the quality of insomnia clinical practice guidelines that involve TCM recommendations and to summarize the certainty of evidence supporting the recommendations, strength, and consistency of recommendations, providing valuable research references for the development of future insomnia guidelines. MATERIALS AND METHODS: We systematically searched PubMed, Web of Science, Embase, CNKI, Wanfang, Chinese Biomedical Literature Database, Chinese Medical Association, Chinese Sleep Research Society, Medsci, Medlive, British National Institute of Health and Clinical Excellence (NICE), and the International Guidelines Collaboration Network (GIN) for clinical practice guidelines on insomnia from inception to March 5, 2023. Four evaluators conducted independent assessments of the quality of the guidelines by employing the AGREE II tool. Subsequently, the guideline recommendations were consolidated and presented as evidence maps. RESULTS: Thirteen clinical practice guidelines addressing insomnia, encompassing 211 recommendations (consisting of 127 evidence-based and 84 expert consensus recommendations), were deemed eligible for inclusion in our analysis. The evaluation results revealed an overall suboptimal quality, with the "scope and purpose" domain achieving the highest score (58.1%), while the "applicability" domain garnered the lowest score (13.0%). Specifically, it was observed that 74.8% (n = 95) of the evidence-based recommendations were supported by evidence of either very low or low certainty, in contrast to the expert consensus recommendations, which accounted for 61.9% (n = 52). We subsequently synthesized 44 recommendations into four evidence maps, focusing on proprietary Chinese medicines, Chinese medicine prescriptions, acupuncture, and massage, respectively. Notably, Chinese herbal remedies and acupuncture exhibited robust support, substantiated by high-certainty evidence, exemplified by interventions such as Xuefu Zhuyu decoction, spleen decoction, body acupuncture, and ear acupuncture, resulting in solid recommendations. Conversely, proprietary Chinese medicines needed more high-certainty evidence, predominantly yielding weak recommendations. As for other therapies, the level of certainty was predominantly categorized as low or very low. Recommendations about magnetic therapy, bathing, and fumigation relied primarily on expert consensus, needing more substantive clinical research evidence, consequently forming weak recommendations. Hot ironing and acupoint injection recommendations were weakly endorsed, primarily based on observational studies. Furthermore, interventions like qigong, gua sha, and moxibustion displayed a relatively limited number of clinical studies, necessitating further exploration to ascertain their efficacy. CONCLUSIONS: Our analysis revealed a need for substantial improvement in the quality of all the included guidelines related to insomnia. Notably, recommendations for Traditional Chinese Medicine (TCM) treatments predominantly rely on low-certainty evidence. This study represents a pioneering effort in the utilization of recommendation mapping to both present and identify existing gaps in the evidence landscape within TCM therapies, thus setting the stage for future research initiatives. The evidence supporting TCM therapy recommendations must be fortified to achieve a more substantial level of recommendation and higher certainty. Consequently, there exists a critical and pressing demand for high-quality clinical investigations dedicated to TCM, with a specific focus on ascertaining its long-term efficacy, safety, and potential side effects in the context of insomnia treatment. These endeavors are poised to establish a robust scientific foundation to inform the development of TCM therapy recommendations within the insomnia guidelines.

A novel multidisciplinary machine learning approach based on clinical, imaging, colonoscopy, and pathology features for distinguishing intestinal tuberculosis from Crohn's disease.

OBJECTIVES: Differentiating intestinal tuberculosis (ITB) from Crohn's disease (CD) remains a diagnostic dilemma. Misdiagnosis carries potential grave implications. We aim to establish a multidisciplinary-based model using machine learning approach for distinguishing ITB from CD. METHODS: Eighty-two patients including 25 patients with ITB and 57 patients with CD were retrospectively recruited (54 in training cohort and 28 in testing cohort). The region of interest (ROI) for the lesion was delineated on magnetic resonance enterography (MRE) and colonoscopy images. Radiomic features were extracted by least absolute shrinkage and selection operator regression. Pathological feature was extracted automatically by deep-learning method. Clinical features were filtered by logistic regression analysis. Diagnostic performance was evaluated by receiver operating characteristic (ROC) curve and decision curve analysis (DCA). Delong's test was applied to compare the efficiency between the multidisciplinary-based model and the other four single-disciplinary-based models. RESULTS: The radiomics model based on MRE features yielded an AUC of 0.87 (95% confidence interval [CI] 0.68-0.96) on the test data set, which was similar to the clinical model (AUC, 0.90 [95% CI 0.71-0.98]) and higher than the colonoscopy radiomics model (AUC, 0.68 [95% CI 0.48-0.84]) and pathology deep-learning model (AUC, 0.70 [95% CI 0.49-0.85]). Multidisciplinary model, integrating 3 clinical, 21 MRE radiomic, 5 colonoscopy radiomic, and 4 pathology deep-learning features, could significantly improve the diagnostic performance (AUC of 0.94, 95% CI 0.78-1.00) on the bases of single-disciplinary-based models. DCA confirmed the clinical utility. CONCLUSIONS: Multidisciplinary-based model integrating clinical, MRE, colonoscopy, and pathology features was useful in distinguishing ITB from CD.

Assessing the Risk of Bias in Randomized Clinical Trials With Large Language Models.

Importance: Large language models (LLMs) may facilitate the labor-intensive process of systematic reviews. However, the exact methods and reliability remain uncertain. Objective: To explore the feasibility and reliability of using LLMs to assess risk of bias (ROB) in randomized clinical trials (RCTs). Design, Setting, and Participants: A survey study was conducted between August 10, 2023, and October 30, 2023. Thirty RCTs were selected from published systematic reviews. Main Outcomes and Measures: A structured prompt was developed to guide ChatGPT (LLM 1) and Claude (LLM 2) in assessing the ROB in these RCTs using a modified version of the Cochrane ROB tool developed by the CLARITY group at McMaster University. Each RCT was assessed twice by both models, and the results were documented. The results were compared with an assessment by 3 experts, which was considered a criterion standard. Correct assessment rates, sensitivity, specificity, and F1 scores were calculated to reflect accuracy, both overall and for each domain of the Cochrane ROB tool; consistent assessment rates and Cohen κ were calculated to gauge consistency; and assessment time was calculated to measure efficiency. Performance between the 2 models was compared using risk differences. Results: Both models demonstrated high correct assessment rates. LLM 1 reached a mean correct assessment rate of 84.5% (95% CI, 81.5%-87.3%), and LLM 2 reached a significantly higher rate of 89.5% (95% CI, 87.0%-91.8%). The risk difference between the 2 models was 0.05 (95% CI, 0.01-0.09). In most domains, domain-specific correct rates were around 80% to 90%; however, sensitivity below 0.80 was observed in domains 1 (random sequence generation), 2 (allocation concealment), and 6 (other concerns). Domains 4 (missing outcome data), 5 (selective outcome reporting), and 6 had F1 scores below 0.50. The consistent rates between the 2 assessments were 84.0% for LLM 1 and 87.3% for LLM 2. LLM 1's κ exceeded 0.80 in 7 and LLM 2's in 8 domains. The mean (SD) time needed for assessment was 77 (16) seconds for LLM 1 and 53 (12) seconds for LLM 2. Conclusions: In this survey study of applying LLMs for ROB assessment, LLM 1 and LLM 2 demonstrated substantial accuracy and consistency in evaluating RCTs, suggesting their potential as supportive tools in systematic review processes.

Dl-3-n-butylphthalide activates Nrf2, inhibits ferritinophagy, and protects MES23.5 dopaminergic neurons from ferroptosis.

Ferroptosis, a newly identified iron-dependent form of cell death, has recently been implicated in the pathogenesis of Parkinson's disease (PD). Dl-3-n-butylphthalide (NBP) attenuates behavioral and cognitive deficits in animal models of PD. However, the potential of NBP to prevent dopaminergic neuron death by suppressing ferroptosis has rarely been explored. In this study, we aimed to investigate the effects of NBP on ferroptosis in erastin-induced dopaminergic neurons (MES23.5 cells) and the underlying mechanisms involved in these effects. Our results demonstrated that erastin significantly decreased viability of MES23.5 dopaminergic neurons in a dose-dependent manner, which was reversible by ferroptosis inhibitors. We further verified that NBP protected erastin-treated MES23.5 cells from death by inhibiting ferroptosis. Erastin increased the mitochondrial membrane density, caused lipid peroxidation, and decreased GPX4 expression in MES23.5 cells, which could be reversed by NBP preconditioning. NBP pretreatment suppressed erastin-induced labile iron accumulation and reactive oxygen species generation. Moreover, we demonstrated that erastin significantly reduced FTH expression, and pre-administration with NBP promoted Nrf2 translocation into the nucleus and increased the protein level of FTH. Additionally, the expression of LC3B-II in MES23.5 cells pretreated with NBP before administration of erastin was lower than that in cells treated with erastin alone. NBP reduced colocalization of FTH and autophagosomes in MES23.5 cells exposed to erastin. Finally, erastin gradually inhibited NCOA4 expression in a time-dependent manner, which was reversible by NBP pretreatment. Taken together, these results indicated that NBP suppressed ferroptosis via regulating FTH expression, which was achieved by promoting Nrf2 nuclear translocation and inhibiting NCOA4-mediated ferritinophagy. As such, NBP may be a promising therapeutic agent for the treatment of neurological diseases associated with ferroptosis.

Ferroptosis contributes to endometrial fibrosis in intrauterine adhesions.

Intrauterine adhesions (IUA), characterized by endometrial fibrosis, is a challenging clinical issue in reproductive medicine. We previously demonstrated that epithelial-mesenchymal transition (EMT) and fibrosis of endometrial stromal cells (HESCs) played a vital role in the development of IUA, but the precise pathogenesis remains elucidated. Ferroptosis has now been recognized as a unique form of oxidative cell death, but whether it is involved in endometrial fibrosis remains unknown. In the present study, we performed an RNA-seq of the endometria from 4 severe IUA patients and 4 normal controls. Enrichment analysis and protein-protein interactions (PPIs) network analysis of differentially expressed genes (DEGs) were conducted. Immunohistochemistry was used to assess ferroptosis levels and cellular localization. The potential role of ferroptosis for IUA was investigated by in vitro and in vivo experiments. Here, we demonstrated that ferroptosis load is increased in IUA endometria. In vitro experiments showed that erastin-induced ferroptosis promoted EMT and fibrosis in endometrial epithelial cells (P < 0.05), but did not lead to pro-fibrotic differentiation in endometrial stromal cells (HESCs). Cell co-culture experiments showed that erastin-stimulated epithelial cell supernatants promoted fibrosis in HESCs (P < 0.05). In vivo experiments suggested that elevation of ferroptosis level in mice by erastin led to mild endometrial EMT and fibrosis. Meanwhile, the ferroptosis inhibitor Fer-1 significantly ameliorated endometrial fibrosis in a dual-injury IUA murine model. Overall, our findings revealed that ferroptosis may serve as a potential therapeutic target for endometrial fibrosis in IUA.