Structural instability and divergence from conserved residues underlie intracellular retention of mammalian odorant receptors.

Mammalian odorant receptors are a diverse and rapidly evolving set of G protein-coupled receptors expressed in olfactory cilia membranes. Most odorant receptors show little to no cell surface expression in nonolfactory cells due to endoplasmic reticulum retention, which has slowed down biochemical studies. Here we provide evidence that structural instability and divergence from conserved residues of individual odorant receptors underlie intracellular retention using a combination of large-scale screening of odorant receptors cell surface expression in heterologous cells, point mutations, structural modeling, and machine learning techniques. We demonstrate the importance of conserved residues by synthesizing consensus odorant receptors that show high levels of cell surface expression similar to conventional G protein-coupled receptors. Furthermore, we associate in silico structural instability with poor cell surface expression using molecular dynamics simulations. We propose an enhanced evolutionary capacitance of olfactory sensory neurons that enable the functional expression of odorant receptors with cryptic mutations.

In vitro activities of a novel antimicrobial peptide isolated from phyllomedusa tomopterna.

Because of the abuse of antibiotics, clinical strains began to become more drug-resistant. Their evolution has long surpassed the speed of us looking for a new generation of antibacterial drugs. Therefore, it is urgent to discover a new antimicrobial substance to alleviate the pressure on conventional antibiotics. Antimicrobial peptides (AMP) are known for their significant activity towards a broad spectrum of bacteria, protozoa, yeasts, filamentous fungi. Here, we report a novel AMP named Dermaseptin-TO. Results demonstrate that Dermaseptin-TO can quickly exhibit antimicrobial activity to bacteria and yeast in a dose-related way. The highest minimum inhibit concentration (MIC) was observed in the E.faecalis group (128 µM). Also, haemolytic outcomes showed no more than 10.65% of red blood cells were affected when in the same concentrations or below. Besides, Dermaseptin-TO also showed anticancer activity at a higher concentration. From the above, evidence proved that Phyllomedusine frog skin secretion is still a rich source that contains novel AMP and Dermaseptin-TO is competent to become an antimicrobial agent, its anticancer activity may broaden the way in basic cancer research. Also, following the same templates in molecular cloning may acquire new AMP classes with potent antimicrobial effects that could widen drug design in new anti-infective drugs.

Triptolide reduces salivary gland damage in a non-obese diabetic mice model of Sjögren's syndrome via JAK/STAT and NF-κB signaling pathways.

Triptolide (TP) has anti-inflammatory and immunosuppressive effects. However, the effect of triptolide on Sjögren's syndrome (SS) is rarely reported. In this paper, we studied the effects of triptolide on non-obese diabetes mice model of SS. In this study, salivary flow rate was measured every two weeks, and autoantibodies levels in the serum were detected. Salivary gland index and spleen index were detected, pathological changes of salivary gland were detected by hematoxylin-eosin staining, inflammatory factors were detected by enzyme linked immunosorbent assay, lymphocytes were detected by flow cytometry, proliferation of T cells and B cells were detected, and related proteins were detected by Western blot. Triptolide increased salivary flow rate and salivary gland index, and decreased spleen gland index. Moreover, triptolide reduced the infiltration of lymphocytes to salivary glands, decreased the level of autoantibodies in serum, and reduced the inflammatory factors in salivary glands and IFN-γ induced salivary gland epithelial cells. Further, triptolide inhibited activator of JAK/STAT pathway and NF-κB pathway. In conclusion, triptolide could inhibit the infiltration of lymphocytes and the expression of inflammatory factors through JAK/STAT pathway and NF-κB pathway. Thus, triptolide may be used as a potential drug to treat SS.

Erratum: Ophiopogonin-B Suppresses Epithelial-mesenchymal Transition in Human Lung Adenocarcinoma Cells via the Linc00668/miR-432-5p/EMT Axis: Erratum.

[This corrects the article DOI: 10.7150/jca.31338.].

Discovery of a Novel Antimicrobial Peptide, Temporin-PKE, from the Skin Secretion of Pelophylax kl. esculentus, and Evaluation of Its Structure-Activity Relationships.

Bacterial resistance against antibiotics has led to increasing numbers of treatment failures, and AMPs are widely accepted as becoming potential alternatives due to their advantages. Temporin-PKE is a novel peptide extracted from the skin secretion of Pelophylax kl. esculentus and it displays a strong activity against Gram-positive bacteria, with an extreme cytotoxicity. Incorporating positively charged residues and introducing D-amino acids were the two main strategies adopted for the modifications. The transformation of the chirality of Ile could reduce haemolytic activity, and an analogue with appropriate D-isoforms could maintain antimicrobial activity and stability. The substitution of hydrophobic residues could bring about more potent and broad-spectrum antimicrobial activities. The analogues with Lys were less harmful to the normal cells and their stabilities remained at similarly high levels compared to temporin-PKE. The optimal number of charges was three, and the replacement on the polar face was a better choice. Temporin-PKE-3K exerted dually efficient functions includingstrong antimicrobial and anticancer activity. This analogue showed a reduced possibility for inducing resistance in MRSA and Klebsiella pneumoniae, a rather strong antimicrobial activity in vivo, and it exhibited the highest therapeutic index such that temporin-PKE-3K has the potential to be developed as a clinical drug.

Kassporin-KS1: A Novel Pentadecapeptide from the Skin Secretion of Kassina senegalensis: Studies on the Structure-Activity Relationships of Site-Specific "Glycine-Lysine" Motif Insertions.

Due to the abuse of traditional antibiotics and the continuous mutation of microbial resistance genes, microbial infections have become serious problems for human health. Therefore, novel antibacterial agents are urgently required, and amphibian antimicrobial peptides (AMP) are among the most interesting potential antibacterial leads. In this research, a novel peptide, named kassporin-KS1 (generically QUB-1641), with moderate antibacterial activity against Gram-positive bacteria, was discovered in the skin secretion of the Senegal running frog, Kassina senegalensis. Using site-specific sequence enrichment with a motif "glycine-lysine" that frequently occurs in ranid frog temporin peptides, a series of QUB-1641 analogues were synthesized, and effects on selected bioactivities were studied. The greatest activity enhancement was obtained when the "glycine-lysine" motif was located at the eighth and ninth position as in QUB-1570.QUB-1570 had a broader antibacterial spectrum than QUB-1641, and was eight-fold more potent. Moreover, QUB-1570 inhibited S. aureus biofilm most effectively, and significantly enhanced the viability of insect larvae infected with S. aureus. When the "glycine-lysine" motif of QUB-1570 was substituted to reduce the helix ratio and positive charge, the antibacterial activities of these synthetic analogues decreased. These data revealed that the "glycine-lysine" motif at positions 8 and 9 had the greatest enhancing effect on the antibacterial properties of QUB-1570 through increasing positive charge and helix content. This research may provide strategies for the site's selective amino acid modification of some natural peptides to achieve the desired enhancement of activity.

Triptolide attenuates inhibition of ankylosing spondylitis-derived mesenchymal stem cells on the osteoclastogenesis through modulating exosomal transfer of circ-0110634.

Background: Ankylosing spondylitis (AS) is featured by chronic inflammation of the sacroiliac joints and spine as well as pathological new bone formation. Osteoclastogenesis is a critical part in the development of bone formation. Circular RNAs (circRNAs) are recent research hotspot in the RNA field while rarely reported in osteoclastogenesis. Methods: AS mesenchymal stem cells (ASMSCs) and healthy donor mesenchymal stem cells (HDMSCs) were co-cultured with peripheral blood mononuclear cells (PBMCs). RT-qPCR was applied to detect the expression level of circ-0110634 in different exosomes. TRAP staining and TRAP activity detection were performed to identify the effect of circ-0110634 overexpression on osteoclastogenesis. Bioinformatics analysis and mechanism investigation were conducted to explore the downstream molecular mechanism of circ-0110634. Results: The effect of ASMSCs on PBMCs osteoclastogenesis is weaker than that of HDMSCs. Circ-0110634 had higher expression in ASMSCs exosomes than HDMSCs exosomes. Circ-0110634 overexpression suppressed the osteoclastogenesis. Circ-0110634 bound to both TNF receptor associated factor 2 (TRAF2) and tumor necrosis factor receptor II (TNFRII). Circ-0110634 also accelerated the dimerization of TRAF2 to induce TRAF2 ubiquitination and degradation. Circ-0110634 repressed the interplay between TRAF2 and TNFRII to inactivate the nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPK) pathways. Triptolide promoted the osteoclastogenesis of ASMSCs exosomes-treated PBMCs via decreasing the exosomal transference of circ-0110634 in a dose-dependent manner. Consistently, triptolide treatment stimulated osteoclastogenesis to alleviate the arthritis of DBA/1 mice through suppressing circ-0110634. Conclusion: Our study confirmed that triptolide targets circ-0110634 to ease the burden of AS patients. The Translational potential of this article: This study suggests triptolide targets circ-0110634 to regulate osteoclastogenesis, which provides a novel potential target in triptolide treatment for AS patients.

Ophiopogonin-B Suppresses Epithelial-mesenchymal Transition in Human Lung Adenocarcinoma Cells via the Linc00668/miR-432-5p/EMT Axis.

Ophiopogonin-B (OP-B) has been reported to suppress metastasis and angiogenesis of adenocarcinoma A549 cells in vitro and in vivo. More and more evidences indicate that inflammatory microenvironment facilitates tumor metastasis. Digital Gene Expression (DGE) analysis of non-small cell lung cancer (NSCLC) cell lines showed that OP-B downregulated the expression of linc00668, which promoted progression of cancer. Herein, we simulated the inflammatory microenvironment by co-culturing A549 cells with LPS-treated THP-1 cells and found that the level of linc00668 increased significantly in the mock group, while OP-B treatment inhibited the level of linc00668 and reversed epithelial-mesenchymal transition (EMT) induced by linc00668. In addition, overexpression of linc00668 in A549 cells suppressed the expression of E-cadherin and induced expression of N-cadherin, while OP-B treatment reversed these changes. Bioinformatic prediction and dual-luciferase reporter gene assay validated that linc00668 sponge miR-432-5p and at last acted on EMT to execute the anti-migration function of A549 cells under inflammatory microenvironment. Taken together, OP-B inhibits metastasis of A549 cells via the linc00668/miR-432-5p/EMT axis.

A 12-week Baduanjin Qigong exercise improves symptoms of ankylosing spondylitis: A randomized controlled trial.

BACKGROUND AND PURPOSE: Therapeutic exercises are considered effective treatments for ankylosing spondylitis(AS). Current study aimed to evaluate efficacy and safety of Baduanjin qigong, a traditional Chinese exercise, for treatment of AS in a pilot RCT setting. MATERIALS AND METHODS: A total of 60 patients were randomly assigned, at a 1:1 ratio, to receive a 12-week Baduanjin qigong training(exercise group) or maintain their current lifestyle(no-treatment group). As primary outcomes, Bath Ankylosing Spondylitis Disease Activity Index(BASDAI) and other AS symptoms were assessed at baseline and end of treatment period. RESULTS: A total of 46 patients completed the study. At the end of treatment period, although total BASDAI scores were not statistically different, reduced scores were observed in the exercise group, compared to no-treatment group, with respect to fatigue(P = 0.03), intensity(P = 0.04) and duration(P = 0.01) of morning stiffness; exercise group also exhibited higher patient global assessment scores(P = 0.04). CONCLUSION: Baduanjin qigong exercise appeared to improve AS symptoms.

Sodium new houttuyfonate suppresses metastasis in NSCLC cells through the Linc00668/miR-147a/slug axis.

BACKGROUND: As most lung cancer patients present with invasive, metastatic disease, it is vital to investigate anti-metastatic treatments for non-small cell lung cancer (NSCLC). Houttuynia cordata is commonly used as a Chinese anticancer medicine in the clinic, and sodium new houttuyfonate (SNH), a main compound of this herb, has long been found to have antibiotic effects, although its anticancer effects have not been investigated. Here, we tried to address this lack of research from the perspective of the competing endogenous RNA (ceRNA) theory. METHODS: The effects of SNH on NSCLC cells were analysed with Cell Counting Kit-8 assays and colony formation assays. In addition, transwell assays and wound healing assays were used to determine the effects of SNH on migration and invasion in NSCLC cells. The levels of key genes and proteins were examined by quantitative real-time PCR, western blotting, immunofluorescence staining and IHC staining. Through transcriptome screening and digital gene expression profiling, Linc00668 was identified to be regulated by SNH. Dual-luciferase reporter assays and RNA immunoprecipitation assays verified the binding efficiency between miR-147a and Linc00668 or Slug. RESULTS: In the present study, SNH regulated NSCLC cells in multiple ways, the most prominent of which was suppressing the expression of Linc00668, which was indicated to promote migration and invasion in NSCLC cells. Functional studies demonstrated that Linc00668 acted as a ceRNA by sponging miR-147a to further regulate Slug mRNA levels, thereby influencing the progression of the epithelial-mesenchymal transition. Consistently, the results of in vivo animal models showed that SNH depressed Linc00668 and suppressed the metastasis of NSCLC. CONCLUSIONS: SNH suppressed metastasis of NSCLC cells and the mechanism may involve with the Linc00668/miR-147a/Slug axis.

Triptolide inhibits proliferation, differentiation and induces apoptosis of osteoblastic MC3T3­E1 cells.

Ankylosing spondylitis (AS) is characterized by the formation of bony spurs. Treatment of the resulting ankylosis, excessive bone formation and associated functional impairment, remain the primary therapeutic aims in research regarding this condition. Triptolide is the primary active component of the perennial vine Tripterygium wilfordii Hook. f., and has previously been demonstrated to exert anti­tumor activities including inhibition of cell growth and the induction of apoptosis, however, the effect of triptolide on osteoblasts remains to be elucidated. In the present study, the MC3T3­E1 mouse osteoblast cell line was treated with differing concentrations of triptolide for various intervals. Cell proliferation was detected using the bromodeoxyuridine assay, cell cycle and apoptosis were measured by flow cytometry, nuclear apoptosis was observed by Hoechst staining and associated proteins were determined via western blot analysis. The cells were then further incubated with osteogenic induction medium supplemented with triptolide for 7 or 12 days and the differentiation to osteoblasts was examined by picrosirius staining, observation of alkaline phosphatase activity and a calcium deposition assay. It was demonstrated that treatment with triptolide significantly inhibited osteoblast proliferation and induced cell cycle arrest and apoptosis of the osteoblasts. Furthermore, treatment with triptolide reduced collagen formation, alkaline phosphatase activity and calcium deposition. The present study demonstrated an inhibitory effect of triptolide on osteoblast proliferation and differentiation, and therefore suggests a potential therapeutic agent for the treatment of AS in the future.