The identification and functional analysis of CircRNAs in endometrial receptivity of mice with polycystic ovary.

The disorders of endometrial receptivity and ovulatory dysfunction are both significant causes of infertility in patients with polycystic ovary syndrome (PCOS). In this study, we investigated the expression profile and functional implications of circular RNAs (circRNAs) in the endometrial receptivity of PCOS-affected mice. Twenty-four female C57BL/6 mice were divided into PCOS and normal control groups. The PCOS group received subcutaneous DHEA treatment, while the control group remained untreated. Gene chip technology was utilized to analyze circRNA expression in endometrial tissues on the fourth day of gestation with subsequent bioinformatics analyses into circRNA functions. Furthermore, endometrial epithelial cells were used to determine represented circRNA functions. Results showed that the PCOS group exhibited 205 differentially expressed circRNAs, with 147 upregulated and 58 downregulated ones. qRT-PCR confirmed differential expression of circRNAs, including circRNA_38548, circRNA_001686, circRNA_38550, and circRNA_27938. Predicted target genes and a circRNA-miRNA-mRNA regulatory network were constructed. Additionally, four circRNAs (circRNA_38548, circRNA_38550, and circRNA_001686) were identified to contribute to abnormal endometrial receptivity by regulating genes such as Lifr, FOXK1, FOXO1, HOXA10, through interactions with miRNAs. Further research is warranted to elucidate the underlying mechanisms involving these circRNAs.

Screening and optimization of a water-soluble near-infrared fluorescent probe for drug-induced liver injury monitoring.

Peroxynitrite (ONOO-) is a potential biomarker of drug-induced liver injury (DILI) and is involved in the process of DILI. Therefore, developing a reliable detection method for ONOO- will greatly contribute to ensuring drug safety and improving treatment efficiency. Here, based on the previous work, two kinds of NIR fluorescence probes PN and SPN were developed with phenyl-hydrazine as the ONOO- recognition group, which based on two fluorophores RN and SRN that are stable to ONOO-. A sensitive NIR probe SPN with good water solubility, low detection limit and good biocompatibility was selected through in vitro spectral property screening. Further experimental results show that there is a good linear relationship between the response intensity of probe SPN to ONOO- and the concentration of ONOO-, and the detection limit can reach 19.7 nM. At the cellular level, probe SPN can achieve a good and specific response to endogenous and exogenous ONOO-. Also, the probe SPN can be used for imaging and detection of DILI in zebrafish level and small animal level, indicating that probe SPN can be used as a powerful tool for diagnosis of DILI and efficacy evaluation of therapeutic drugs.

Discovery and characterization of hydroxylysine O-glycosylation in an engineered IL-2 fusion protein.

In the present study, an engineered interleukin-2 (IL-2) fusion protein consisting of an anti-human serum albumin nanobody linked by ASTKG and a (G4S)2 linker to IL-2 was constructed. Liquid chromatography-mass spectrometry (LC-MS) characterization was performed on the intact molecule and at the peptide level. The LC-MS molecular mass analysis for the engineered fusion protein showed the appearance of unreported +340 Da peaks, apart from the expected O-glycosylation-related peaks in the IL-2 domain. Through a combination analysis of a K120R mutated molecule (The lysine at the position of 120 was mutated to arginine while the rest amino acid sequence remain unchanged), the possibility of a non-cleaved valine-histidine-serine signal peptide was ruled out and the presence of hydroxylysine (HyK) O-glycosylation in the ASTKG linker was confirmed. HyK O-glycosylation have been reported in other proteins such as collagen, which occurs in the conserved Gly-Xaa-HyK motif and is catalyzed by lysyl hydroxylase-3 complex. The present study showed high similar conserved motif of HyK-O-glycosylation in collagen, implying the HyK O-glycosylation in the engineered IL-2 possibly was catalyzed by the Chinese hamster ovary homolog of enzymes promoting HyK O-glycosylation in collagen. Bioactivity testing results revealed that HyK-O-glycosylation had no obvious effect on the in vitro activity of engineered IL-2. Our study is the first to report HyK-O-glycosylation modifications in therapeutic proteins through LC-MS characterization and in vitro activity analysis, which expands the scope of post-translational modification knowledge of therapeutic proteins.

Screening diagnostic markers for acute myeloid leukemia based on bioinformatics analysis.

Background: An in-depth understanding of the key molecules and associated mechanisms involved in acute myeloid leukemia (AML) carcinogenesis, proliferation, and relapse is critical. This provides a basis for disease screening, early diagnosis, and development of effective treatment strategies and prognosis. Methods: We downloaded AML transcription data sets from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Differentially expressed genes (DEGs) were screened by R software and limma packages. Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed on DEGs by public databases. In the DEG set, a random forest algorithm was used to identify characteristic genes of AML. The receiver operator characteristic (ROC) curve was used to evaluate the diagnostic efficacy of selected characteristic genes, which provided clues for the discovery of early diagnostic markers. The Estimate score was calculated using the Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) algorithm. Spearman's correlation test was used to explore the correlation between characteristic genes and Estimate Score, which provided clues for clarifying the potential pathogenic mechanism of key genes. Results: A total of 1,494 DEGs were identified from AML samples and normal samples, among which 1,181 genes were upregulated and 313 genes were downregulated in AML. There were 2 genes with a mean decrease Gini >2, namely, CDC20 and ESM1, respectively. The ROC curve showed that the area under the curve (AUC) of CDC20 was 0.966, and the 95% confidence interval (CI) was (0.939 to 0.987) (P<0.001). The AUC of ESM1 was 0.905, and 95% CI: 0.849 to 0.953 (P<0.001). Correlation analysis showed that CDC20 expression was negatively correlated with Estimate Score (R=-0.21, P=0.0036) in AML. The expression of ESM1 was negatively correlated with Estimate Score (R=-0.57, P<0.001). Conclusions: The genes CDC20 and ESM1 were identified as AML characteristic genes by random forest algorithm. Both CDC20 and ESM1 have good diagnostic efficacy for AML. They may play a carcinogenic role by promoting tumor cell proliferation and inhibiting immune cell chemotaxis, which are potential biological markers.

Nomogram incorporating ultrasonic markers of endometrial receptivity to determine the embryo-endometrial synchrony after in vitro fertilization.

Background: A successful pregnancy using in vitro fertilization and embryo transfer (IVF-ET) requires a receptive endometrium, good-quality embryos, and a synchronized embryo-endometrial dialogue. Although embryo quality and endometrial receptivity (ER) have been fully assessed to exclude substandard conditions, the probability of successful ET is relatively low. Currently, embryo-endometrial synchrony is considered to be a possible explanation, because delayed, advanced, or narrowed window of implantation (WOI) may lead to ET failure. Objective: This study aims to establish a nomogram incorporating a series of ultrasonic ER markers on the day before implantation to assess the embryo-endometrial synchrony, which may contribute to the improvement of clinical pregnancy outcomes. Methods: Totally 583 women with 1135 complete IVF cycles were retrospectively analyzed. Among them, 357 women with 698 cycles and 226 women with 437 cycles were assigned to the training and validation cohorts, respectively. Ultrasonic ER markers obtained on the day before implantation were collected for analyses. In the training cohort, the screened correlates of clinical pregnancy failure were utilized to develop a nomogram for determining whether an infertile woman is suitable for the ET next day. This model was validated both in the training and validation cohorts. Results: Spiral artery (SA) resistance index (RI), vascularisation index (VI), and flow index (FI) were independently associated with the ET failure (all P < 0.05). They were served as the components of the developed nomogram to visualize the likelihood of implantation failure in IVF-ET. This model was validated to present good discrimination and calibration, and obtained clinical net benefits both in the training and validation cohorts. Conclusion: We developed a nomogram that included SA-RI, VI, and FI on the day before implantation. It may assist physicians to identify patients with displaced WOI, thus avoiding meaningless ET prior to implantation.

Different Chinese herbal medicine therapy for idiopathic thrombocytopenic purpura: A protocol for systematic review and Bayesian network meta-analysis.

BACKGROUND: Idiopathic thrombocytopenic purpura (ITP) is a common immune system and blood system disease in clinical practice, and it is a hemorrhagic disease caused by immune factors causing platelet destruction and decreasing number of platelets. There is currently no effective treatment plan for ITP. At this stage, glucocorticoid and gamma globulin are mostly used in the treatment of ITP, and some patients use splenectomy to achieve therapeutic purposes, but the various treatment methods are inadequate. At this stage, a large number of randomized controlled studies have reported that Chinese herbal medicine has achieved good curative effect in the treatment of ITP. However, due to the variety of Chinese herbal medicine, there has been no evidence of the effectiveness and safety of Chinese herbal medicine in the treatment of ITP. Because of the above reasons, this study uses the network meta-analysis method based on Bayesian method to compare the clinical efficacy and safety of different kinds of Chinese herbal medicine in the treatment of ITP through direct and indirect comparison, in order to provide evidence-based medical support for the treatment of ITP with Chinese herbal medicine. METHODS: This study uses the method of combining free words with theme words, and using computer to retrieve PubMed, EMbase, The Cochrane Library, WANFANG Database, CNKI, and VIP Database, etc, to collect the randomized control studies on Chinese herbal medicine in the treatment of ITP. The retrieval time is from the establishment of each database to January 2021, and the retrieval languages are Chinese and English. Two researchers independently read the title, abstract and full text of the article to determine whether it is included in the literature; In the event of a disagreement, a third researcher will join the discussion to resolve the disagreement; For the literature that lacks information, trying to contact the original author of the document to supplement it. The literature quality evaluation carried out by using the Stata 14.0 software to draw network and funnel plots, in accordance with the quality evaluation criteria of version 5.1.0 of the Cochrane System Evaluation Manual. Statistical analysis is performed by using ADDIS 1.16.8 software based on the Bayesian model. RESULTS: This study will compare the clinical efficacy and safety of different types of Chinese herbal medicine in the treatment of idiopathic thrombocytopenic purpura through the method of network meta-analysis, and rank the different types of Chinese herbal medicine according to their effectiveness, and the results will be published in a peer-reviewed, high-quality academic journal. CONCLUSION: This study will find effective and safe Chinese herbal medicine for clinical treatment of ITP from the perspective of evidence-based medicine, and benefit more ITP patients.

A near-infrared fluorescent probe with large Stokes shift for visualizing and monitoring mitochondrial viscosity in live cells and inflammatory tissues.

Mitochondria are cellular energy factory, having an essential role in cellular metabolism. Furthermore, abnormal changes in mitochondrial viscosity have been confirmed to be closely related to many diseases. Therefore, the development of probe that responsive to mitochondrial viscosity and its application in mitochondrial viscosity measurement is considered to be a new tool for understanding diseases. In this paper, a mitochondrial viscosity probe (DICB) with a large Stokes shift (214-253 nm) was designed and synthesized by modifying the structure of the carbazole fluorophore. The probe DICB has a favorable responsive to viscosity in the near-infrared (NIR) region (703 nm). In the water-glycerol system (0.893 cP -945 cP), the fluorescence intensity of DICB at 703 nm has a 74 times increase; in the range of 5.041 cP-856.0 cp, it has a well linear fitting relationship. Meantime, the probe has excellent sensitivity to viscosity. The probe (DICB) has been confirmed to be able to detect changes of mitochondrial viscosity in cell models induced by nystatin, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and lipopolysaccharide (LPS); it has also been validated that DICB can be used in the process of autophagy to monitor mitochondrial viscosity. More importantly, DICB can be applied to the detection of abnormal mitochondrial viscosity in inflammatory tissues at the biological level. The outstanding characteristics of DICB for mitochondrial viscosity detection are not only of great importance to the development of viscosity probes, but also provides a universal strategy to study the relationship between inflammatory and mitochondrial viscosity.

A visible and near-infrared dual-fluorescent probe for discrimination between Cys/Hcy and GSH and its application in bioimaging.

Biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play important roles in various physiological and pathological processes, and due to their similar structures and reaction activities, it is still challenging to simultaneously discriminate between GSH and Cys/Hcy in vivo. Hence, a novel fluorescent probe for simultaneously discriminating GSH and Cys/Hcy in biological samples is highly desirable. Herein, we presented two enhanced fluorescent probes (Cy1 and Cy2) with doubly-activated dual emission for sensitive and discriminative detection of Cys/Hcy and GSH. The probes were constructed with IR-780 and NBD linked via an ether bond, which responds to GSH with near infrared (NIR) emission at 810 nm (λex = 720 nm) and Cys/Hcy with visible green emission at 550 nm (λex = 470 nm). The probe Cy2 is operable in human serum samples, thus holding promise for its diagnosis-related application. Notably, the results of fluorescence microscopy imaging indicated that Cy2 is suitable for visualization of exogenous and endogenous biothiols in living cells. Furthermore, desirable results were obtained when the probe Cy2 was applied for bioimaging in tumor-bearing mice and acute liver injury (ALI) mice models, which revealed encouraging clinical values of this probe.

Spectrum of gene mutations identified by targeted next-generation sequencing in Chinese leukemia patients.

BACKGROUND: Despite targeted sequencing have identified several mutations for leukemia, there is still a limit of mutation screening for Chinese leukemia. Here, we used targeted next-generation sequencing for testing the mutation patterns of Chinese leukemia patients. METHODS: We performed targeted sequencing of 504 tumor-related genes in 109 leukemia samples to identify single-nucleotide variants (SNVs) and insertions and deletions (INDELs). Pathogenic variants were assessed based on the American College of Medical Genetics and Genomics (ACMG) guidelines. The functional impact of pathogenic genes was explored through gene ontology (GO), pathway analysis, and protein-protein interaction network in silico. RESULTS: We identified a total of 4,655 SNVs and 614 INDELs in 419 genes, in which PDE4DIP, NOTCH2, FANCA, BCR, and ROS1 emerged as the highly mutated genes. Of note, we were the first to demonstrate an association of PDE4DIP mutation and leukemia. Based on ACMG guidelines, 39 pathogenic and likely pathogenic mutations in 27 genes were found. GO annotation showed that the biological process including gland development, leukocyte differentiation, respiratory system development, myeloid leukocyte differentiation, mesenchymal to epithelial transition, and so on were involved. CONCLUSION: Our study provided a map of gene mutations in Chinese patients with leukemia and gave insights into the molecular pathogenesis of leukemia.

Bioinformatics analysis of differentially expressed genes on alopecia.

The molecular mechanism of alopecia areata (AA) is still elusive and here we utilized bioinformatics methods to analyze AA-related differentially expressed genes. In this study, GSE45512 and GSE45513 were downloaded from the NCBI sub-database Gene Expression Omnibus (GEO). The gene expressions of AA and normal samples were analyzed using the R package limma, which showed significant differences between AA and normal samples in two species. These genes were subject to functional annotation and protein interaction networks. At the same time, gene set enrichment analysis was conducted for all differentially expressed genes. The study revealed that a total of 225 differentially expressed genes were screened from human AA samples, and a total of 337 differentially expressed genes were screened from spontaneous AA skin samples in C3H/HeJ mice. There are 23 differentially expressed genes in the two species. GO and protein interaction network analysis shown gene enrichment in immune-related functions, and these proteins interact with each other. Gene set enrichment analysis showed that differential genes from both species were significantly enriched to chemokine signaling pathways, cytokine-cytokine receptor interactions, staphylococcus aureus infection, and antigen processing and presentation. Moreover, the human down-regulated differential gene not only maps to the alopecia in human phenotype ontology, but also maps to the pathologically relevant phenotype of the skin appendage. In brief, 23 significant differentially expressed genes were screened out coexisting in AA human and mouse by bioinformatics methods. In addition, the result demonstrated that AA is closely related to the immune process and skin appendage lesions. These results provide new ideas for the diagnosis and treatment of AA.

microRNA-22 can regulate expression of the long non-coding RNA MEG3 in acute myeloid leukemia.

AIM: Acute myeloid leukemia (AML) is the most common blood tumor with poor prognosis. At present, the research found that the pathogenesis of AML is related to many factors, such as recurrent somatic mutations and gene expression and epigenetic changes, however, the molecular mechanism of AML is still unclear. Long non-coding RNA MEG3 is a newly found tumor suppressor and plays a very important role in the regulation of a variety of tumor formation and progression. Studies found that the MEG3 expression was significantly decreased in AML. However, to date, it is not clear the cause of its abnormal expression. Therefore, the molecular mechanism of AML is urgently needed to study the molecular mechanism of AML. METHODS: The different expression level of MEG3, TET2, miR-22-3p, miR-22-5p in AML was detected by real-time quantification PCR. MEG3, TET2, miR-22-3p, miR-22-3p expression cell pools in K562 cells was used to interfering and TET2, MEG3 TET2, relations with miR-22-3p, miR-22-5p. The effect of AML cell on proliferation was evaluated by TET2 lower expression. RESULTS: 1. The lower expression of MEG3 and TET2 in AML cell lines was detected by RT-qPCR. 2. The stable MEG3, TET2 overexpression cell pools in K562 cells was successful established. 3. After transfection, MTT assay revealed that cell growth was significantly increased in AML cell lines transfected with TET2 compared with controls. CONCLUSIONS: Our findings suggested that MEG3 is significantly down regulated in AML cell lines.

Effects and safety of GnRH-a as a luteal support in women undertaking assisted reproductive technology procedures: follow-up results for pregnancy, delivery, and neonates.

PURPOSE: To investigate the effects and safety of gonadotropin releasing hormone analogue (GnRH-a) as an addition to progesterone luteal support in women who underwent in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) and achieved a clinical pregnancy. METHODS: A retrospective analysis was conducted on 214 patients who underwent IVF/ICSI-ET procedures with standard long mid-luteal protocol, of which 123 received GnRH-a-free protocol and 91 received GnRH-a-added protocol. The patients' pregnancy and delivery course, and their neonates' status at birth and growth/development after birth were statistically compared. RESULTS: There was no significant difference between both study groups regarding embryo risks and maternal complications during early pregnancy. as well as fetal risks during the middle and late stages and neonate risks during birth, except that the twin pregnancies of the GnRH-a-added group had a considerably greater male/female ratio, and a significantly higher rate of premature delivery and low birth weight than those of the GnRH-a-free group. In addition, there was no significant difference in neonate risks within 2 years after birth between both cohorts. CONCLUSION: With precautions taken to control the number of implanted embryos and reduce the incidence of twinning pregnancy, the addition of GnRH-a to luteal support is relatively safe and effective.

[Analysis of the follow-up results concerning pregnancy, delivery and infants after assisted reproductive technique with GnRH-a for luteal support].

OBJECTIVE: To investigate the results of follow-up visits of pregnancy course, delivery and infants of women who got clinically pregnant by assisted reproductive technique after gonadotropin-releasing hormone agonist (GnRH-a) added for luteal support, and to analyse the influence of adding GnRH-a in luteal support on the safety of mother and infant. METHODS: A retrospective analysis was carried out on the medical record from 215 patients who got clinically pregnant after luteal phase long regimen fresh-cycle transfer was operated. According to the differences in luteal support methods, the patients were assigned to Group A (124 patients, progesterone+dydrogesterone group), Group B (91 patients, GnRH-a added group). The patients' pregnancy course, delivery time, and the growth and development of infants within 1-2 years were followed up. RESULTS: (1) There was no obvious difference between Group A and Group B in terms of the abortion ratio during the early pregnancy (8.1%, 12.1%), the rate of abortion villous deformity (50.0%, 9.1%), the rate of heterotopic pregnancy (10.5%, 5.5%) and rate of twin pregnancy (19.4%, 28.6%; all P>0.05). (2) Compared to group A, during the middle and late pregnancy of single or twin pregnancy in Group B , there was no obvious difference in the rate of fetal chromosomal abnormality, organ malformation incidence, late abortion rate and stillbirth rate (all P>0.05). (3) As to childbirth, in the case of twin pregnancy, there was a higher rate of premature delivery (60.0%, 39.1%; P=0.041), as well as rate of lower birth weight of newborn (56.0%, 34.8%; P=0.037) in group B. (4) The statistics on general growth and development as well as infantile common diseases within 2 years after birth indicated that there was no obvious difference between the two groups in single birth and twin birth subgroup (all P>0.05). CONCLUSION: On the basis of controlling of implanted embryos and reducing the occurrence of twins, GnRH-a luteal support maybe relatively safe and effective.

MiR-20a regulates the PRKG1 gene by targeting its coding region in pulmonary arterial smooth muscle cells.

Chronic hypoxia triggers pulmonary vascular remodeling, which is associated with de-differentiation of pulmonary artery smooth muscle cells (PASMC). Here, we show that miR-20a expression is up-regulated in response to hypoxia in both mouse and human PASMC. We also observed that miR-20a represses the protein kinase, cGMP-dependent, type I (PRKG1) gene and we identified two crucial miR-20a binding sites within the coding region of PRKG1. Functional studies showed that miR-20a promotes the proliferation and migration of human PASMC, whereas it inhibits their differentiation. In summary, we provided a possible mechanism by which hypoxia results in decreased PRKG1 expression and in the phenotypic switching of PASMC.

[Rapid detection of Pseudomonas aernginosa by the fluorescence quantitative TaqMan PCR assay targetting ETA gene].

Pseudomonas aernginosa (PA) is one of the most universal pathogens in clinical diagnosis, and conventional detection assay has many disadvantages. In this research, a pair of specific primers and a TaqMan fluorescent probe were designed in the conservative region of ETA gene by the method of bioinformatics analysis, the detection method for PA was successfully developed. Different gradient concentrations of PA DNA and various pathogen DNA were amplified by fluorescence quantitative PCR (FQ-PCR) to confirm the specificity and sensitivity of the developed method. Results showed that the developed detection assay is more sensible and specific by comparison to the conventional FQ-PCR method, and it is valuable for research and application prospects.