Highly multiplex PCR assays by coupling the 5'-flap endonuclease activity of Taq DNA polymerase and molecular beacon reporters.

Real-time PCR is the most utilized nucleic acid testing tool in clinical settings. However, the number of targets detectable per reaction are restricted by current modes. Here, we describe a single-step, multiplex approach capable of detecting dozens of targets per reaction in a real-time PCR thermal cycler. The approach, termed MeltArray, utilizes the 5'-flap endonuclease activity of Taq DNA polymerase to cleave a mediator probe into a mediator primer that can bind to a molecular beacon reporter, which allows for the extension of multiple mediator primers to produce a series of fluorescent hybrids of different melting temperatures unique to each target. Using multiple molecular beacon reporters labeled with different fluorophores, the overall number of targets is equal to the number of the reporters multiplied by that of mediator primers per reporter. The use of MeltArray was explored in various scenarios, including in a 20-plex assay that detects human Y chromosome microdeletions, a 62-plex assay that determines Escherichia coli serovars, a 24-plex assay that simultaneously identifies and quantitates respiratory pathogens, and a minisequencing assay that identifies KRAS mutations, and all of these different assays were validated with clinical samples. MeltArray approach should find widespread use in clinical settings owing to its combined merits of multiplicity, versatility, simplicity, and accessibility.

25(OH)D3 improves granulosa cell proliferation and IVF pregnancy outcomes in patients with endometriosis by increasing G2M+S phase cells.

BACKGROUND: The 25-hydroxyvitamin D3 (25 (OH) D3) is crucial for follicular development. This study aimed to investigate the relationship between the level of 25 (OH) D3 in endometriosis patients, pregnancy outcomes of in vitro fertilization (IVF), and the underlying mechanism. METHODS: The 25 (OH) D3 levels in serum and follicular Fluid (FF) samples were detected using enzyme-linked immunosorbent assay (ELISA). Clinical features and pregnancy outcomes of endometriosis patients were also compared between the deficient group (< 20 ug/ml) and the adequate group (≥ 20 ug/ml). The effects of 25 (OH) D3 on the proliferation and cell cycle of human ovarian granulosa cells were respectively detected by CCK-8 assay and flow cytometry (FCM). The differentially expressed genes (DEGs) in granulosa cells of endometriosis and tubal infertility patients were screened from GEO database. The effects of 25 (OH) D3 on the expressions of CDKN2D, PPARA, TGFB2 and THBD were determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. RESULTS: The levels of 25 (OH) D3 in serum and FF samples were decreased in endometriosis patients. The deficient group had fewer embryos that can be transferred, lower quality embryos and lower clinical pregnancy rates. Adequate 25 (OH) D3 levels in FF samples was a protective factor for live birth outcome in endometriosis patients. 25 (OH) D3 enhanced the proliferation capacity of granulosa cells (the concentration of 10 nM was the most significant) and increased the proportion of G2M + S phase cells. The expression of CDKN2D was decreased and TGFB2 and THBD were significantly upregulated. CONCLUSIONS: 25 (OH) D3 deficiency may be associated with poor IVF pregnancy outcomes in endometriosis patients. 25 (OH) D3 promotes ovarian granulosa cell proliferation by promoting the ability of cells to divide, and may accelerate cell cycle progression by up-regulating THBD and down-regulating CDKN2D expression.

Expression and significance of SOX B1 genes in glioblastoma multiforme patients.

The overall survival of glioblastoma multiforme (GBM) patients remains poor. To improve patient outcomes, effective diagnostic and prognostic biomarkers for GBM are needed. In this study, we first applied bioinformatic analyses to identify biomarkers for GBM, focusing on SOX (sex-determining region on the Y chromosome (SRY)-related high mobility group (HMG) box) B1 family members. The ONCOMINE, GEPIA, LinkedOmics and CCLE databases were used to assess mRNA expression levels of the SOX B1 family members in different cancers and normal tissue. Further bioinformatic analysis was performed using the ONCOMINE database in combination with the LinkedOmics data set to identify the prognostic value of SOX B1 family members for GBM. We found mRNA expression levels of all tested SOX B1 genes were significantly increased in GBM. In the LinkedOmics database, increased expression of SOX3 indicated a better overall survival. In GEPIA databases, increased expression of all SOX B1 family members suggested an improved overall survival, but none of them were statistically different. Then, Transwell assays and wound healing were employed to evaluate the motility and invasive captivity of U251 cells when silencing SOX2 and SOX3. We found exogenous inhibition of SOX2 appeared to reduce the migration and invasion of U251 cells in vitro. Collectively, our research suggested that SOX2 might serve as a cancer-promoting gene to identify high-risk GBM patients, and SOX3 had the potential to be a prognostic biomarker for GBM patients.

MgO-loaded tubular ceramic membrane with spatial nanoconfinement for enhanced catalytic ozonation in refractory wastewater treatment.

Heterogeneous catalytic ozonation (HCO) enables the destruction of organic pollutants in wastewater via oxidation by powerful hydroxyl radicals (·OH). However, the availability of short-lived ·OH in aqueous bulk is low in practical treatment scenarios due to mass transfer limitations and quenching of water constituents. Herein, we overcome these challenges by loading MgO catalysts inside the pores of a tubular ceramic membrane (denoted as CCM) to confine ·OH within the nanopores and achieve efficient pollutant removal. When the pore size of the membrane was reduced from 1000 to 50 nm, the removal of ibuprofen (IBU) by CCM was increased from 49.6 % to 90.2 % due to the enhancement of ·OH enrichment in the nanospace. In addition, the CCM exhibited high catalytic activity in the presence of co-existing ions and over a wide pH range, as well as good self-cleaning ability in treating secondary wastewater. The experimental results revealed that ·OH were the dominant reactive oxygen species (ROS) in pollutant degradation, while surface hydroxyl groups were active sites for the generation of ·OH via ozone decomposition. This work provides a promising strategy to enhance the utilization of ·OH in HCO for the efficient degradation of organic pollutants in wastewater under spatial confinement.

Corrigendum: Sestrin2 protects against hypoxic nerve injury by regulating mitophagy through SESN2/AMPK pathway.

[This corrects the article DOI: 10.3389/fmolb.2023.1266243.].

The utility of diffusion-weighted imaging for differentiation of phyllodes tumor from fibroadenoma and breast cancer.

Objective: To evaluate the utility of apparent diffusion coefficient (ADC) values for differentiating breast tumors. Methods: The medical records of 17 patients with phyllodes tumor [PT; circular regions of interest (ROI-cs) n = 171], 74 patients with fibroadenomas (FAs; ROI-cs, n = 94), and 57 patients with breast cancers (BCs; ROI-cs, n = 104) confirmed by surgical pathology were retrospectively reviewed. Results: There were significant differences between PTs, FAs, and BCs in ADCmean, ADCmax, and ADCmin values. The cutoff ADCmean for differentiating PTs from FAs was 1.435 × 10-3 mm2/s, PTs from BCs was 1.100 × 10-3 mm2/s, and FAs from BCs was 0.925 × 10-3 mm2/s. There were significant differences between benign PTs, borderline PTs, and malignant PTs in ADCmean, ADCmax, and ADCmin values. The cutoff ADCmean for differentiating benign PTs from borderline PTs was 1.215 × 10-3 mm2/s, and borderline PTs from malignant PTs was 1.665 × 10-3 mm2/s. Conclusion: DWI provides quantitative information that can help distinguish breast tumors.

LAMC2 regulates proliferation, migration, and invasion mediated by the Pl3K/AKT/mTOR pathway in oral.

Background: Oral squamous cell carcinoma (OSCC) is a common malignant tumor. Recently, Laminin Gamma 2 (LAMC2) has been shown to be abnormally expressed in OSCC; however, how LAMC2 signaling contributes to the occurrence and development of OSCC and the role of autophagy in OSCC has not been fully explored. This study aimed to analyze the role and mechanism of LAMC2 signaling in OSCC and the involvement of autophagy in OSCC. Methods: To explore the mechanism by which LAMC2 is highly expressed in OSCC, we used small interfering RNA (siRNA) to knock down LAMC2 to further observe the changes in the signaling pathway. Furthermore, we used cell proliferation assays, Transwell invasion assays, and wound-healing assays to observe the changes in OSCC proliferation, invasion, and metastasis. RFP-LC3 was used to detect the level of autophagy intensity. A cell line-derived xenograft (CDX) model was used to detect the effect of LAMC2 on tumor growth in vivo. Results: This study found that the level of autophagy was correlated with the biological behavior of OSCC. The downregulation of LAMC2 activated autophagy and inhibited OSCC proliferation, invasion, and metastasis via inhibiting the PI3K/AKT/mTOR pathway. Moreover, autophagy has a dual effect on OSCC, and the synergistic downregulation of LAMC2 and autophagy can inhibit OSCC metastasis, invasion, and proliferation via the PI3K/AKT/mTOR pathway. Conclusions: LAMC2 interacts with autophagy to regulate OSCC metastasis, invasion, and proliferation via the PI3K/AKT/mTOR pathway. LAMC2 down-regulation can synergistically modulate autophagy to inhibit OSCC migration, invasion, and proliferation.

Exogenous Antigen Upregulation Empowers Antibody Targeted Nanochemotherapy of Leukemia.

Acute myeloid leukemia (AML) is afflicted by a high-mortality rate and few treatment options. The lack of specific surface antigens severely hampers the development of targeted therapeutics and cell therapy. Here, it is shown that exogenous all-trans retinoic acid (ATRA) mediates selective and transient CD38 upregulation on leukemia cells by up to 20-fold, which enables high-efficiency targeted nanochemotherapy of leukemia with daratumumab antibody-directed polymersomal vincristine sulfate (DPV). Strikingly, treatment of two CD38-low expressing AML orthotopic models with ATRA and DPV portfolio strategies effectively eliminates circulating leukemia cells and leukemia invasion into bone marrow and organs, leading to exceptional survival benefits with 20-40% of mice becoming leukemia-free. The combination of exogenous CD38 upregulation and antibody-directed nanotherapeutics provides a unique and powerful targeted therapy for leukemia.

Sestrin2 protects against hypoxic nerve injury by regulating mitophagy through SESN2/AMPK pathway.

Hypoxia induced by high altitude can lead to severe neurological dysfunction. Mitophagy is known to play a crucial role in hypoxic nerve injury. However, the regulatory mechanism of mitophagy during this injury remains unclear. Recent studies have highlighted the role of Sestrin2 (SESN2), an evolutionarily conserved stress-inducible protein against acute hypoxia. Our study demonstrated that hypoxia treatment increased SESN2 expression and activated mitophagy in PC12 cells. Furthermore, the knock-out of Sesn2 gene led to a significant increase in mitochondrial membrane potential and ATP concentrations, which protected the PC12 cells from hypoxic injury. Although the AMPK/mTOR pathway was significantly altered under hypoxia, it does not seem to participate in mitophagy regulation. Instead, our data suggest that the mitophagy receptor FUNDC1 plays a vital role in hypoxia-induced mitophagy. Moreover, SESN2 may function through synergistic regulation with other pathways, such as SESN2/AMPK, to mediate cellular adaptation to hypoxia, including the regulation of mitophagy in neuron cells. Therefore, SESN2 plays a critical role in regulating neural cell response to hypoxia. These findings offer valuable insights into the underlying molecular mechanisms governing the regulation of mitophagy under hypoxia and further highlight the potential of SESN2 as a promising therapeutic target for hypoxic nerve injury.

Clinical outcomes of personalized frozen-thawed embryo transfer timing for patients with recurrent implantation failure.

Background: Recurrent implantation failure (RIF) is a critical problem for assisted reproduction technology. High-quality embryos and the synchronization endometrium both have great significance. How to get the optimal endometrial receptivity is a challenge for implantation and pregnancy of infertile patients with RIF. The objective of this study is to investigate personalized protocol of frozen-thawed embryo transfer (FET) cycles, and its effect on clinical outcomes in patients with RIF. Methods: We chose 91 RIF patients from January 2017 to June 2019 in the Reproductive Medicine Center of the First Hospital of Lanzhou University. A total of 100 FET cycles were undertaken with a gonadotropin-releasing hormone agonist (GnRH-agonist) protocol combined with hormone replacement therapy (HRT) for endometrial preparation. The patients were divided into two groups: the routine group (cleavage embryo transferred at day 3 after luteal support) included 48 cycles; the personalized group included 52 cycles with delayed endometrial trigger and luteal support (the time of embryo transfer depended on the level of serum hormone and endometrial thickness). Results: The data showed the personalized group had longer time for endometrial preparation. On the day of embryo transfer, serum progesterone (P) and the E2/P ratio was significantly different compared with the routine group (P<0.05). The clinical pregnancy rate in the routine group was 35.42% (17/48) and 59.62% (31/52) in the personalized group (P<0.05). The abortion rate was not significantly different. Conclusions: For women with RIF, personalized timing for transfer of FET resulted in a higher clinical pregnancy rate compared with routine protocol.

[Effects of three Polyphenolic compounds on the intestinal flora of mice exposed simulated intermittent plateau hypoxia].

OBJECTIVE: To investigate the protective effects of three Polyphenolic compounds on intestinal microbial communities in mice exposed intermittent plateau hypoxia. METHODS: In this study, 60 healthy male Balb/c mice were randomly divided into plain control group, plateau control group, primary anthocyanin intervention group, quercetin intervention group and resveratrol intervention group, 12 mice in each group. Primary anthocyanin, quercetin and resveratrol were administrated by gavage at the doses of 50, 100 and 20 mg/kg in pharmacological intervention group, respectively. After exposure of the mice to simulation plateau-condition for 30 days, the serum samples were collected for DAO testing, sterile feces were collected in mice, and the diversity and genus level of the mouse gut bacteria were detected by using 16S rRNA technology. Ileum tissue was fixed and stained with HE. RESULTS: HE staining showed that the plateau control group had significant damage to the intestinal tissue structure compared to the plain control group, and the serum DAO concentration was increased (P＜0.05), but there was no statistical difference in the abundance and diversity of intestinal flora species. Contrast to simulated intermittent plateau hypoxia group, the structure of the intestine tissue and the level of DAO in the quercetin intervention group and resveratrol intervention group were improved(P＜0.05), the abundance and α diversity of the intestinal flora were decreased, the relative abundance of Bacteroidetes was reduced(P＜0.05), and the Firmicutes was increased. Concomitantly, significant decreases in relative abundance were observed for Corynebacterium glutamicum and Lactobacillus reuteri(P＜ 0.05). CONCLUSION: Quercetin and resveratrol showed some degree of protection to mice intestinal microbial communities, and increased the diversity and the abundance of the dominant flora and inhibited the growth of conditional pathogenic bacteria.

[Determination and identification of acrylamide residues in poly-acrylamide by ultra-performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometry].

A new method for the direct determination of acrylamide residues in poly-acrylamide (PAM) by ultra-performance liquid chromatography (UPLC)-quadrupole electrostatic field orbitrap high-resolution mass spectrometry with the Hypercarb column was developed. With being diluted by water, the PAM sample was dissolved well by high-speed stirring. After filtering through a 0.22 µm aqueous phase microporous membrane, the sample was analyzed by high-resolution mass spectrometry directly. Chromatographic analysis was carried out using a Hypercarb column, distilled water and methanol applied as the mobile phases. In addition, the column temperature was strictly set at 20℃. The sample was determined in the mode of parallel reaction monitoring (PRM). Accurate mass-to-charge ratio extraction of the target compound ion and its fragment ion were utilized with elemental composition analysis and isotope distribution of the fragment ion to further improve the accuracy of qualitative and quantitative analysis. Meanwhile, the fragmentation pattern of acrylamide was acquired. The calibration curve showed a good linearity in the range of 2-50 µg/L with the correlation coefficient of 0.9998. The limit of detection (LOD) was 1.5 µg/kg, satisfying the detection demands. The recoveries of the acrylamide were in the range of 101.3%-107.1%. The relative standard deviations (RSD) were 3.1%-4.1%. The applications indicated that the mass percentages of AM in PAM were in the range of 0-0.43%. The proposed method is easy, fast, sensitive and suitable for the determination and confirmation of acrylamide residues in PAM.