An efficient and universal In silico screening strategy for acquisition of high-affinity Aptamer and its application in analytical utility.

Numerous aptamers against various targets have been identified through the technology of systematic evolution of ligands by exponential enrichment (SELEX), but the affinity of these aptamers are often insufficient due to the limitations of SELEX. Therefore, a more rational in silico screening strategy (ISS) was developed for efficient screening of high affinity aptamers, which took shape complementarity and thermodynamic stability into consideration. Neuron specific enolase (NSE), a tumor marker, was selected as the target molecule. In the screening process, three aptamer candidates with good shape complementarity, lower ΔG values, and higher ZDOCK scores were produced. The dissociation constant (Kd) of these candidates to NSE was determined to be 10.13 nM, 14.82 nM, and 2.76 nM, respectively. Each of them exhibited higher affinity to NSE than the parent aptamer (Kd = 23.83 nM). Finally, an antibody-free fluorescence aptasensor assay, based on the aptamer with the highest affinity, P-5C8G, was conducted, resulting in a limit of detection (LOD) value of 1.8 nM, which was much lower than the parental aptamer (P, LOD = 12.6 nM). The proposed ISS approach provided an efficient and universal strategy to improve the aptamer to have a high affinity and good analytical utility.

miR-27a-5p alleviates periodontal inflammation by targeting phosphatase and tensin homolog deleted on chromosome ten.

INTRODUCTION: MicroRNAs (miRNAs), a type of non-coding RNA, have been demonstrated to be essential posttranscriptional modulators in oral diseases and inflammatory responses. However, the specific role of miR-27a-5p in periodontitis requires further investigation. In this study, we used both cellular and animal models to determine how miR-27a-5p affects the pathogenesis of periodontitis and its associated biological functions. METHODS: Quantitative real-time polymerase chain reaction and western blotting were used to analyze the expression of cytokines, phosphatase and tensin homolog deleted on chromosome ten (PTEN), and miR-27a-5p transcription. Investigation of alveolar bone resorption and inflammation of the periodontium in ligature-induced periodontitis in mice was performed using micro-computed tomography (micro-CT), hematoxylin-eosin (HE) staining, and tartrate-resistant acid phosphatase (TRAP) staining. The binding of miR-27a-5p and PTEN was predicted using the TargetScan database and experimentally confirmed using dual luciferase reporter gene assays. RESULTS: The inflamed gingiva showed lower levels of miR-27a-5p. Macrophages from miR-27a-5p-/- mice produced much higher quantities of pro-inflammatory cytokines owing to the stimulation of Porphyromonas gingivalis lipopolysaccharide, and miR-27a-5p-/- mice with ligature-induced periodontitis also exhibited more severe alveolar bone resorption and damage to the periodontium. Target validation assays identified PTEN as a direct target of bona. Blocking PTEN expression partially reduced inflammation, both in vitro and in vivo. CONCLUSIONS: miR-27a-5p alleviated the inflammatory response in periodontitis by targeting PTEN.

Resveratrol improves follicular development of PCOS rats via regulating glycolysis pathway and targeting SIRT1.

Polycystic ovary syndrome (PCOS) is a disease characterized by metabolic disorders. This study aimed to examine the effects of resveratrol treatment on ovulation in the PCOS rat model. Quantitative real-time PCR and immunohistochemistry were used to determine the mRNA and protein expression levels. TNUEL assay was used to evaluate cell apoptosis in ovary. The metabolites were evaluated by liquid chromatography with tandem mass spectrometry. Resveratrol alleviated disrupted estrous cycle and improved granular cell layers, and reversed the decreased proliferation and increased cell apoptosis of granulosa cells in the ovarian tissues of PCOS rats. Resveratrol restored the changes in the mRNA expression levels in the rate-limiting genes of glycolysis in the PCOS ovary. The expression of lactate dehydrogenase A (LDH-A), pyruvate kinase isozyme M2 (PKM2), and sirtuin 1 (SIRT1) was significantly downregulated in ovarian tissues of the PCOS rats; while the resveratrol treatment significantly increased the expression of LDH-A, PKM2, and SIRT1 in the ovarian tissues of PCOS rats. Collectively, the protective effects of resveratrol in the PCOS rats may be associated with the regulation of glycolysis-related mediators including PKM2, LDH-A, and SIRT1. Resveratrol may represent a good candidate in alleviating the development of PCOS.

Posttranslational modifications in pathogenesis of PCOS.

Polycystic ovary syndrome (PCOS) is a lifelong reproductive, metabolic, and psychiatric disorder that affects 5-18% of women, which is associated with a significantly increased lifetime risk of concomitant diseases, including type 2 diabetes, psychiatric disorders, and gynecological cancers. Posttranslational modifications (PTMs) play an important role in changes in protein function and are necessary to maintain cellular viability and biological processes, thus their maladjustment can lead to disease. Growing evidence suggests the association between PCOS and posttranslational modifications. This article mainly reviews the research status of phosphorylation, methylation, acetylation, and ubiquitination, as well as their roles and molecular mechanisms in the development of PCOS. In addition, we briefly summarize research and clinical trials of PCOS therapy to advance our understanding of agents that can be used to target phosphorylated, methylated, acetylated, and ubiquitinated PTM types. It provides not only ideas for future research on the mechanism of PCOS but also ideas for PCOS treatments with therapeutic potential.

Characterization of IL-10-producing regulatory B cells in thymoma.

BACKGROUND: Regulatory B cells (Bregs) are a subset of B cells that secrete interleukin 10 (IL-10) and play a vital role in suppressing the immune response. The aim of this study was to evaluate the proportion of Bregs in patients with thymoma. METHODS: The proportions of subgroups of Bregs in 23 patients with thymoma and 15 healthy controls were detected by flow cytometry. The serum IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α levels of the subjects were measured using a cytometric bead array (CBA). RESULTS: The proportions of circulating IL-10+ B cells, IL-10+CD24hiCD38hi Bregs, and IL-10+CD24hiCD27+ Bregs and the serum IL-10 level were significantly higher in patients with thymoma than in the control group and were negatively correlated with the Karnofsky Performance Scale (KPS) score. The serum levels of cytokines IL-2, IL-6, IFN-γ, and TNF-α were higher and serum IL-17A level was lower in patients with thymoma. Patients with advanced-stage thymoma exhibited significantly higher proportions of IL-10-producing Bregs and a higher serum IL-10 level. After tumour resection, the frequency of circulating IL-10+CD24hiCD38hi Bregs and the serum IL-10 level were significantly decreased in patients with thymoma. The serum IL-10 levels exhibited the best accuracy in assessing the risk of thymoma occurrence in this study. CONCLUSIONS: The expression of IL-10 produced by Bregs is increased in patients with thymoma, particularly those with advanced-stage disease, which may suggest that Bregs are involved in the pathogenesis and progression of thymoma.

Follicular fluid-derived exosomal miR-143-3p/miR-155-5p regulate follicular dysplasia by modulating glycolysis in granulosa cells in polycystic ovary syndrome.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is characterized by follicular dysplasia. An insufficient glycolysis-derived energy supply of granulosa cells (GCs) is an important cause of follicular dysplasia in PCOS. Follicular fluid (FF) exosomal microRNAs (miRNAs) have been proven to regulate the function of GCs. In this study, exosomes extracted from clinical FF samples were used for transcriptome sequencing (RNA-seq) analysis, and a human ovarian granulocyte tumour cell line (KGN cells) was used for in vitro mechanistic studies. METHODS AND RESULTS: In FF exosomal RNA-seq analysis, a decrease in glycolysis-related pathways was identified as an important feature of the PCOS group, and the differentially expressed miR-143-3p and miR-155-5p may be regulatory factors of glycolysis. By determining the effects of miR-143-3p and miR-155-5p on hexokinase (HK) 2, pyruvate kinase muscle isozyme M2 (PKM2), lactate dehydrogenase A (LDHA), pyruvate, lactate and apoptosis in KGN cells, we found that upregulated miR-143-3p expression in exosomes from the PCOS group inhibited glycolysis in KGN cells; knockdown of miR-143-3p significantly alleviated the decrease in glycolysis in KGN cells in PCOS. MiR-155-5p silencing attenuated glycolytic activation in KGN cells; overexpression of miR-155-5p significantly promoted glycolysis in KGN cells in PCOS. In this study, HK2 was found to be the mediator of miR-143-3p and miR-155-5p in FF-derived exosome-mediated regulation of glycolysis in KGN cells. Reduced glycolysis accelerated apoptosis of KGN cells, which mediated follicular dysplasia through ATP, lactate and apoptotic pathways. CONCLUSIONS: In conclusion, these results indicate that miR-143-3p and miR-155-5p in FF-derived exosomes antagonistically regulate glycolytic-mediated follicular dysplasia of GCs in PCOS. Video Abstract.

Discovery and characterization of amentoflavone as a naturally occurring inhibitor against the bile salt hydrolase produced by Lactobacillus salivarius.

Bile salt hydrolases (BSHs), a group of cysteine-hydrolases produced by gut microbes, play a crucial role in the hydrolysis of glycine- or taurine-conjugated bile acids and have been validated as key targets to modulate bile acid metabolism. This study aims to discover one or more efficacious inhibitors against a BSH produced by Lactobacillus salivarius (lsBSH) from natural products and to characterize the mechanism of the newly identified BSH inhibitor(s). Following screening of the inhibition potentials of more than 100 natural compounds against lsBSH, amentoflavone (AMF), a naturally occurring biflavone isolated from various medicinal plants, was discovered to be an efficacious BSH inhibitor (IC50 = 0.34 µM). Further investigation showed that AMF could strongly inhibit the lsBSH-catalyzed hydrolytic reaction in living gut microbes. Inhibition kinetic analyses demonstrated that AMF reversibly inhibited the lsBSH-catalyzed hydrolytic reaction in a mixed-inhibition manner, with an apparent Ki value of 0.65 µM. Fluorescence quenching assays suggested that AMF could quench the fluorescence of lsBSH via a static quenching procedure. Docking simulations suggested that AMF could be fitted into lsBSH at two distinct ligand-binding sites, mainly via hydrophobic interactions and hydrogen bonding, which explained well the mixed inhibition mode of this agent. Animal tests showed that the hydrolytic activities of BSHs in mice feces could be significantly blocked by AMF. In summary, this study reports that AMF is a strong, naturally occurring inhibitor of lsBSH, which offers a promising lead compound to develop novel agents for modulating bile acid metabolism in the host via targeting BSHs.

Design, synthesis and biological evaluation of indanone-chalcone hybrids as potent and selective hCES2A inhibitors.

Human carboxylesterase 2 (hCES2A), one of the major serine hydrolases distributed in the small intestine, plays a crucial role in hydrolysis of ester-bearing drugs. Accumulating evidence has indicated that hCES2A inhibitor therapy can modulate the pharmacokinetic and toxicological profiles of some important hCES2A-substrate drugs, such as the anticancer agent CPT-11. Herein, a series of indanone-chalcone hybrids are designed and synthesized to find potent and highly selective hCES2A inhibitors. Inhibition assays demonstrated that most indanone-chalcone hybrids displayed strong to moderate hCES2A inhibition activities. Structure-hCES2A inhibition activity relationship studies showed that introduction of a hydroxyl at the C4' site and introduction of an N-alkyl group at the C6 site were beneficial for hCES2A inhibition. Particularly, B7 (an N-alkylated 1-indanone-chalcone hybrid) exhibited the most potent inhibition on hCES2A and excellent specificity (this agent could not inhibit other human esterases including hCES1A and butyrylcholinesterase). Inhibition kinetic analyses demonstrated that B7 potently inhibited hCES2A-mediated FD hydrolysis in a mixed inhibition manner, with a calculated Ki value of 0.068 µM. Furthermore, B7 was capable of inhibiting intracellular hCES2A in living cells and displayed good metabolic stability. Collectively, our findings show that indanone-chalcone hybrids are good choices for the development of hCES2A inhibitors, while B7 is a promising candidate for the development of novel anti-diarrhea agents to ameliorate irinotecan-induced intestinal toxicity.

Combined use of Diane-35 and metformin improves the ovulation in the PCOS rat model possibly via regulating glycolysis pathway.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disease with unknown pathogenesis. However, the treatment of Diane-35 combined with metformin can improve the endocrine and ovulation of PCOS. In this study, we investigated the effects of Diane-35 combined with metformin (DM) treatment on ovulation and glucose metabolism in a PCOS rat model. METHODS: Sprague Dawley rats were divided into 3 groups, control group, model group (PCOS group) and Diane-35 combined with metformin (PCOS + DM group). The mRNA expression levels were determined by qRT-PCR. The hormone levels were determined by enzyme-linked immunosorbent assay. Immunostaining detected the protein levels of lactate dehydrogenase A (LDH-A), pyruvate kinase isozyme M2 (PKM2) and sirtuin 1 (SIRT1) in the ovarian tissues. TNUEL assay was performed to determine cell apoptosis in the PCOS rats. The metabolites in the ovarian tissues were analyzed by liquid chromatography with tandem mass spectrometry. RESULTS: PCOS rats showed an increased in body weight, levels of luteinizing hormone and testosterone and insulin resistance, which was significantly attenuated by the DM treatment. The DM treatment improved disrupted estrous cycle and increased the granulosa cells of the ovary in the PCOS rats. The decreased proliferation and increased cell apoptosis of granulosa cells in the ovarian tissues of PCOS rats were significantly reversed by the DM treatment. The analysis of metabolics revealed that ATP and lactate levels were significantly decreased in PCOS rats, which was recovered by the DM treatment. Furthermore, the expression of LDH-A, PKM2 and SIRT1 was significantly down-regulated in ovarian tissues of the PCOS rats; while the DM treatment significantly increased the expression of LDH-A, PKM2 and SIRT1 in the ovarian tissues of the PCOS rats. CONCLUSION: In conclusion, our study demonstrated that Diane-35 plus metformin treatment improved the pathological changes in the PCOS rats. Further studies suggest that Diane-35 plus metformin can improve the energy metabolism of the ovary via regulating the glycolysis pathway. The mechanistic studies indicated that the therapeutic effects of Diane-35 plus metformin treatment in the PCOS rats may be associated with the regulation of glycolysis-related mediators including PKM2, LDH-A and SIRT1.

Dendrobium nobile Lindl. polysaccharides improve follicular development in PCOS rats.

Polycystic ovary syndrome (PCOS) is the most typical and common metabolic abnormalities in women of reproductive age. This study examined the protective effects of Dendrobium nobile Lindl. polysaccharides (DNLP) on ovarian follicular development in letrozole-induced PCOS rats and explored the underlying molecular mechanisms. The PCOS rats showed the increased body weight, serum testosterone and luteinizing hormone levels and insulin resistance. DNLP treatment reduced the body weight, serum testosterone level and insulin resistance, but failed to affect luteinizing hormone level in the PCOS rats. DNLP treatment recovered disrupted estrous cycle in the PCOS rats. DNLP treatment decreased antral follicles and increased the thickness of the granular cell layer. DNLP treatment increased the PCNA mRNA and protein expression levels in the PCOS ovarian tissues, and inhibited cell apoptosis in the PCOS ovarian tissues via regulating apoptosis-related proteins including Bax, Bcl-2 and caspase-3. In summary, this study demonstrated the protective effects of DNLP on the ovaries in the letrozole-induced PCOS rat model. DNLP exerted its protective effects via improving follicular development and inhibiting apoptosis of ovarian granular cells in PCOS rats. This study will provide experimental basis for the future clinical application of DNLP in the treatment of PCOS.

Nanoparticle-Mediated Cavitation via CO2 Laser Impacting on Water: Concentration Effect, Temperature Visualization, and Core-Shell Structures.

By taking advantage of seeded polymer nanoparticles and strong photo energy absorption, we report CO2 laser impacting on water to produce cavitation at the air/water interface. Using a high-speed camera, three regimes (no cavitation, cavitation, and pseudo-cavitation) are identified within a broad range of nanoparticles concentration and size. The underlying correlation among cavitation, nanoparticles and temperature is revealed by the direct observation of spatiotemporal evolution of temperature using a thermal cameral. These findings indicate that nanoparticles not only act as preexisted nuclei to promote nucleation for cavitation, but also likely affect temperature to change the nucleation rate as well. Moreover, by exploiting a compound hexane/water interface, a novel core-shell cavitation is demonstrated. This approach might be utilized to attain and control cavitations by choosing nanoparticles and designing interfaces while operating at a lower laser intensity, for versatile technological applications in material science and medical surgery.

Genome-wide identification of GhAAI genes reveals that GhAAI66 triggers a phase transition to induce early flowering.

Plants undergo a phase transition from vegetative to reproductive development that triggers floral induction. Genes containing an AAI (α-amylase inhibitor) domain form a large gene family, but there have been no comprehensive analyses of this gene family in any plant species. Here, we identified 336 AAI genes from nine plant species including122 AAI genes in cotton (Gossypium hirsutum). The AAI gene family has evolutionarily conserved amino acid residues throughout the plant kingdom. Phylogenetic analysis classified AAI genes into five major clades with significant polyploidization and showing effects of genome duplication. Our study identified 42 paralogous and 216 orthologous gene pairs resulting from segmental and whole-genome duplication, respectively, demonstrating significant contributions of gene duplication to expansion of the cotton AAI gene family. Further, GhAAI66 was preferentially expressed in flower tissue and as responses to phytohormone treatments. Ectopic expression of GhAAI66 in Arabidopsis and silencing in cotton revealed that GhAAI66 triggers a phase transition to induce early flowering. Further, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis of RNA sequencing data and qRT-PCR (quantitative reverse transcription-PCR) analysis indicated that GhAAI66 integrates multiple flower signaling pathways including gibberellin, jasmonic acid, and floral integrators to trigger an early flowering cascade in Arabidopsis. Therefore, characterization of the AAI family provides invaluable insights for improving cotton breeding.

Metal centered oxidation or ligand centered oxidation of metal dithiolene? Spectral, electrochemical and structural studies on a nickel-4-pyridine-1,2-dithiolate system.

The complex Et(4)N[Ni(4-pedt)(2)] (1) (4-pedt = 1-(pyridine-4-yl) ethylene-1,2-dithiolate) was synthesized to investigate the behaviour of metal dithiolene compounds upon protonation and oxidation by absorption spectroscopy, electrochemistry and structural analyses and to further understand the electronic states of the dithiolene compounds. It is unexpected that the 915 nm NIR transition band is not shifted when H(+) is added, and it is only affected (blue-shifted) when the compound is oxidized. All the evidence of electronic spectra indicates that the NIR band is relevant to the central [Ni(edt)(2)] moiety (edt = ethylenedithiolate), not the behaviour of individual Ni ions or ligands. It is also not the band of intermolecular interaction of a dimer. The moderately intense band appearing at 655 nm upon protonation is assigned to the intramolecular charge-transfer band between the [Ni(edt)(2)] moiety and the pyridine. The redox potentials of the metal dithiolene are sensitive to the protonation of the pyridyl group. The structures of monocationic complex and the protonated compounds [Ni(4-Hpedt)(2)]·ClO(4)·H(2)O (2) and [Ni(4-Hpedt)(2)]·PhSO(3)·2DMF (3) were characterized by single crystal X-ray determination. The structural data demonstrate that the oxidation of the monoanionic dithiolene complex to neutral does not change the Ni-S bond distances obviously, which further indicates that the process is not only the metal centered oxidation.