Reducing the mass and decreasing the bioavailability of heavy mental from organic wastes treated by black soldier fly larvae.

Black soldier fly larvae (BSFL), Hermetia illucens L., are widely used to reduce the mass of various wastes. However, the potential metal tolerance mechanisms during periods of waste bioconversion by BSFL remain largely unknown. To further reveal the mechanisms, BSFL were used to treat the agricultural organic wastes, including pig manure (PM), cow manure (COM), spent mushroom substrate (SMS), and wet distiller grains (WDG). After these individual and combined waste(s) were treated by BSFL, we investigated the waste reduction rates and evaluated the responses of BSFL gut microbes to heavy metals of agricultural organic wastes. Additionally, the colloidal particles of residual wastes were characterized by combing energy dispersive X-ray (EDX) spectroscopy, Size potential, Zeta potential, and excitation-emission matrix (EEM) spectroscopy. Results indicated that the waste reduction rates were up to 74% in COM+WDG and 69% in WDG, most of heavy metals (e.g., Zn and Co) from organic wastes were not accumulated in the bodies of mature larvae after treatment. Further, results obtained from the prediction of gene function on the basis of 16 S rRNA data revealed that the presence of multi-resistance genes in the gut of BSFL can help the larvae resist Zn and/or Co stress. In addition, the drug sensitivity test implied that BSFL5_L and BSFL6_L from BSFL gut bacterial strains have multi-resistance to Co and Zn. Additionally, EDX results revealed that the colloidal particles in five waste residues after BSFL treatment are mainly consisted of Fe, Ca and Si, which can capture heavy metals (e.g., Cu, Mn). Results from EEM spectroscopy and PARAFAC showed that tryptophan-like and humic-like accumulatively account for 56%- 68% of all components. Importantly, these two components could strongly bind the metal elements and form colloidal particles with high stability, and therefore reduce the heavy metal pollution of agricultural organic wastes. Our findings offered an environment-friendly method to treat agricultural organic wastes, which would be far-reaching influence to our environment.

Upregulation of miRNA-26a Enhances the Apoptosis of Cerebral Neurons by Targeting EphA2 and Inhibiting the MAPK Pathway.

Neural tube defects (NTDs) constitute the second most common congenital malformation of the central nervous system. The pathogenesis of NTDs is not entirely clear. In recent years, microRNAs (miRNAs) have become a hot spot in genetic and developmental biology research. The present study aimed to explore the potential role of miRNA-26a in NTDs and the underlying pathogenesis thereof. First, we found significantly increased miRNA-26a expression in fetuses with NTDs (p < 0.0001), which significantly downregulated EphA2 and ERK1 mRNA and protein expression levels in fetuses with NTDs compared to normal controls (p < 0.01). In addition, a dual-luciferase reporter assay showed that miR-26a negatively regulated EphA2 by directly binding with the 3'-untranslated region of EphA2. Second, the upregulation of miRNA-26a expression increased caspase 3 and 9 protein expression levels (p < 0.01) and decreased EphA2 mRNA and protein expression levels (p < 0.01), as well as ERK1 and SRF protein expression levels (p < 0.01) in mouse neural stem cells (NE-4C) and human astroblastoma cells (U87MG). Furthermore, the upregulation of miRNA-26a inhibited cell proliferation and enhanced apoptosis of NE-4C and U87MG cells (p < 0.05). Similar results were observed with the MAPK inhibitor PD98059 (p < 0.01). These results suggest that miR-26a targets EphA2, modulates phosphorylation of the MAPK/ERK (MEK) pathway, regulates SRF, and participates in regulating nervous cell proliferation and apoptosis. Dysregulation of the aforementioned mechanism may be involved in the pathogenesis of NTDs.

Protocol for the Detection of Organoid-Initiating Cell Activity in Patient-Derived Single Fallopian Tube Epithelial Cells.

Identification of serous tubal intraepithelial carcinomas (STIC) in the fallopian tubes of women who are carriers of germ line pathogenic variants in tubo-ovarian cancer predisposition genes (i.e., BRCA1 and BRCA2) has led to the hypothesis that many high-grade serous carcinomas (HGSC) arise from the fimbria of the fallopian tube. However, the primitive (stem and progenitor) tubal epithelial cells that give rise to STIC and HGSC have not been defined. Further, as putative HGSC precursors are discovered at salpingectomy, the natural history of such lesions is truncated at diagnosis. Thus, living cultures of human fallopian tubes suitable for experimental studies are needed to define and characterize the cellular origin of HGSCs and thereby advance the discovery of improved methods to assess risk, develop effective early detection tests and identify novel prevention approaches. Accordingly, patient-derived tissue-organoids and isolated epithelial stem cell derived-organoids generated from average and high-risk patients are vital resources to understand the developmental biology of aging fallopian tubes and pathogenesis of HGSCs. With a vision to boost HGSC prevention research, we have established state-of-the-art protocols for the collection, processing, storage, distribution, and management of fallopian tube tissues. Here we describe the protocol for preparing these organoids, with emphasis on the key steps that require meticulous attention to achieve success.

Lnc-RNA LINC01279 induces endometriosis via targeting of HOXA10.

AIM: To explore the regulatory role and molecular mechanism of lncRNA-LINC01279 in endometriosis (EMs). METHODS: Between September 2018 and July 2019, 20 EMs patients and 20 healthy subjects were recruited to detect the expression of lncRNA-LINC01279 in EMs and in normal endometrium via qRT-PCR. Autograft was used to establish EMs models on Spraque-Dawley (SD) rats, which was followed by taking volume measurements of EMs endometrium and observing pathological changes in the morphology of EMs via hematoxylin and eosin (H&E) staining. The qRT-PCR technique was further carried out to determine mRNA expression of lncRNA-LINC01279 and HOXA10 in the serum of EMs rats and LINC01279 shRNA-transfected rats, while the protein expression of HOXA10 was determined using a Western blot. RESULTS: EMs patients presented with upregulation of lncRNA-LINC01279 and downregulation of HOXA10 (p < 0.01 or 0.001). Online predictions further revealed that lncRNA-LINC01279 regulated the expression of HOXA10 via miRNA-135b. In EMs models, it was observed that there were a significantly enlarged endometrium and poor pathological morphology, significant upregulation of lncRNA-LINC01279, and downregulation of miR-135b and HOXA10 in serum (p < 0.05, 0.01 or 0.001). In the lncRNA-LINC01279 shRNA group, EMs rats, following treatment, had a sharp decrease in the volume of EMs endometrium, and an improvement in pathological morphology, while lncRNA-LINC01279 was downregulated, with upregulation of miR-135b and HOXA10 (p < 0.05, 0.01 or 0.001). CONCLUSION: LncRNA-LINC01279, by the mechanism of targeting miR-135b, has the potential to downregulate the expression of HOXA10, and therefore, can promote the development and progression of EMs.

The Influence Mechanism of Abnormal Immunophilin FKBP52 on the Expression Levels of PR-A and PR-B in Endometriosis Based on Endometrial Stromal Cell Model in Vitro.

As a chaperone protein of progesterone receptor (PR), FK-506 Binding Protein 52 (FKBP52) can enhance the activity of PR, but the mechanism of FKBP52 affecting PR expression levels is difficult to clarify. Here, we report a novel in vitro model of ectopic endometrial stromal cells (ESCM) established through the primary culture method of endometrial stromal cells, which is used to study the details of relationship between FKBP52 abnormality and PR expression level in endometriosis (Ems). At the same time, the clinical study of the relationship between FKBP52 and PR expression levels in endometriosis patients was used to verify our conclusions. The results showed that the expression levels of PR-A mRNA and protein in endometriosis are positively correlated with FKBP52 and the abnormality of FKBP52 leads to the decrease of PR-B mRNA and protein expression. When FKBP52 was deleted or reduced, the expression levels of m RNA and protein of PR-A and PR-B have decreased leading to the proliferation of ectopic endometrium cells (ESC) and the occurrence of endometriosis, which is consistent with the expression levels of clinical endometriosis patients and fully confirms our conclusions and reliability of the model, and has great guiding significance for the research of Ems disease occurrence mechanism and clinical treatment.

Increased miRNA-518b inhibits trophoblast migration and angiogenesis by targeting EGR1 in early embryonic arrest†.

Evidence indicates that microRNAs (miRNAs) play essential roles in early embryonic development. The miRNA-518 family is a special biomarker of the placenta, and miRNA-518b is abnormally expressed in placental tissue in preeclampsia. Early growth response protein 1 (EGR1), a zinc finger transcriptional factor, plays an essential role in regulating cell differentiation, angiogenesis, and migration. Moreover, earlier studies have shown that EGR1 protein plays a key role in implantation. However, little is known about the role of miR-518b and EGR1 on early embryonic arrest (EEA) in humans. In our study, increased miR-518b along with decreased EGR1 was found in human villus tissues with EEA. Furthermore, we demonstrated by luciferase assay that miR-518b is a direct regulator of EGR1. After comparing the effect of silencing EGR1, vascular endothelial growth factor (VEGF) individually, and EGR1/VEGF in combination, we found that EGR1 can inhibit migration and angiogenesis of HTR-8 SVneo cells by decreasing the VEGF expression. Hypoxia plays an initial role in early embryonic development, and we found that hypoxia reduces the expression of miR-518b and increases the expression of EGR1 and VEGF to facilitate migration and angiogenesis in a hypoxic model of HTR-8/SVneo cell line. Our findings provide new insights into the role of miR-518b in EEA and implicate the potential application of miR-518b in the diagnosis and development of intervention for EEA.

High-throughput transcriptome-Seq and small RNA-Seq reveal novel functional genes and microRNAs for early embryonic arrest in humans.

Early Embryonic Arrest (EEA) is one of the major causes of female infertility. Genetic factors including specific genes and miRNAs may play pivotal roles on EEA. However, it is not well defined what genes and micro RNAs participate the pathophysiological alterations of EEA. In this work, we compared the Transcriptome -Seq and microRNA profiles from three pairs of villi (three EEA patients and three normal pregnancy, NP). We first confirmed the array data by qPCR with ten randomly selected differentially expressed genes and ten differentially expressed miRNAs in villi from 20 EEA and 20 NP controls. We next applied Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analysis and found that these differentially expressed genes enriched in the PI3K-Akt signaling pathway, Jak-STAT signaling pathway, MAPK signaling pathway, Complement and coagulation cascades, Hypertrophic cardiomyopathy (HCM), Dilated cardiomyopathy (DCM). Interestingly, hsa-miR-6515-5p and its target genes NLRP3, UGP2 may regulate the Immune system and carbohydrate metabolism. Hsa-miRNA 518 and its target gene EGR1 may regulate cell proliferation, angiogenesis, and cell apoptosis to impact early embryonic development. Moreover, novel-m0045-5p and its target gene RMDN3 may regulate microtubule formation on the development of EEA. Our research provides novel biomarkers for EEA and establishes a foundation for further study of the mechanism of EEA.

Early missed abortion is associated with villous angiogenesis via the HIF-1α/VEGF signaling pathway.

PURPOSE: To analyze the effects of the hypoxia-inducible factor 1-alpha (HIF-1α)/vascular endothelial growth factor (VEGF) signaling pathway on villous angiogenesis in early missed abortion. METHODS: Immunohistochemical assays were performed to detect the expression of micro-vessel density (MVD), HIF-1α, and VEGF in villous tissue samples from 30 missed abortions and 30 elective abortions in early pregnancy. We further analyzed the correlation between HIF-1α/VEGF and MVD. HTR8/SVneo cells were cultured under hypoxic (1%) or normoxic (20%) conditions, tube formation was investigated, and protein and mRNA level of HIF-1α/VEGF were determined using western blot and qRT-PCR. Finally, HIF-1α was knocked down with siRNA introduced into HTR8/SVneo cell line under hypoxia, and HIF-1α/VEGF expression and HTR8/SVneo tube formation were investigated. RESULTS: The expression of HIF-1α, VEGF, and MVD was lower in the missed abortion than in the elective abortion group. Correlational analysis showed that the expression of HIF-1α and VEGF was positively correlated with MVD in both groups. The levels of HIF-1α/VEGF mRNA and protein in HTR8/SVneo cells were significantly enhanced under hypoxia. HIF-1α knockdown with siRNA inhibited HIF-1α/VEGF mRNA and protein levels of HTR8/SVneo cells induced by hypoxia. Tube formation of HTR8/SVneo cells was significantly enhanced in hypoxic culture and was inhibited by HIF-1α knockdown with siRNA. CONCLUSIONS: Our results reveal a novel role for HIF-1α/VEGF in regulating villous angiogenesis in early pregnancy and suggest that it may be a novel biomarker for missed abortion.