Xiaoxing Luo

Exendin-4, a Glucagon-Like Peptide-1 Receptor Agonist, Prevents Osteopenia by Promoting Bone Formation and Suppressing Bone Resorption in Aged Ovariectomized Rats

Osteoporosis mainly affects postmenopausal women and older men. Gastrointestinal hormones released after meal ingestion, such as glucose‐dependent insulinotropic peptide (GIP) and glucagon‐like peptide (GLP)‐2, have been shown to regulate bone turnover. However, whether GLP‐1, another important gastrointestinal hormone, and its analogues also have antiosteoporotic effects, especially in aged postmenopausal situation, has not been confirmed. In the present study, we evaluated the effects of the GLP‐1 receptor agonist exendin‐4 on ovariectomy (OVX)‐induced osteoporosis in old rats. Twelve‐month‐old female Sprague‐Dawley rats were subjected to OVX, and exendin‐4 was administrated 4 weeks after the surgery and lasted for 16 weeks. Bone characters and related serum and gene biomarkers were analyzed. Sixteen weeks of treatment with exendin‐4 slowed down body weight gain by decreasing fat mass and prevented the loss of bone mass in old OVX rats. Exendin‐4 also enhanced bone strength and prevented the deterioration of trabecular microarchitecture. Moreover, exendin‐4 decreased the urinary deoxypyridinoline (DPD)/creatinine ratio and serum C‐terminal cross‐linked telopeptides of type I collagen (CTX‐I) and increased serum alkaline phosphatase (ALP), osteocalcin (OC), and N‐terminal propeptide of type 1 procollagen (P1NP) levels, key biochemical markers of bone turnover. Interestingly, gene expression results further showed that exendin‐4 not only inhibited bone resorption by increasing the osteoprotegerin (OPG)/receptor activator of NF‐κB ligand (RANKL) ratio, but also promoted bone formation by increasing the expression of OC, Col1, Runx2, and ALP, which exhibited dual regulatory effects on bone turnover as compared with previous antiosteoporotic agents. In conclusion, these findings demonstrated for the first time the antiosteoporotic effects of exendin‐4 in old OVX rats and that it might be a potential candidate for treatment of aged postmenopausal osteoporosis.

Antisense inhibition of gene expression and growth in gram-negative bacteria by cell-penetrating peptide conjugates of peptide nucleic acids targeted to rpoD gene.

Gram-negative bacteria (GNB) cause common and severe hospital- and community-acquired infections with a high incidence of multidrug resistance (MDR) and mortality. The emergence and spread of MDR-GNB strains limit therapeutic options and highlight the need to develop new therapeutic strategies. In this study, the peptide (RXR)(4)XB- and (KFF)(3)K-conjugated peptide nucleic acids (PPNAs) were developed to target rpoD, which encodes an RNA polymerase primary σ(70) that is thought to be essential for bacterial growth. Their antimicrobial activities were tested against different clinical isolates of MDR-GNB in vitro and in infection models. The (RXR)(4)XB- and (KFF)(3)K- conjugated PNAs were bactericidal against different strains of MDR-GNB in concentration-dependent and sequence-selective manner, whereas a PPNA with a scrambled base sequence had no effect on growth. Among tested PPNAs, (RXR)(4)XB conjugate PPNA06 showed more potent and broad spectrum inhibition in multidrug-resistant Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, and Shigella flexneri in vitro and in vivo. The results were associated with suppression of rpoD mRNA and σ(70) expression, as well as σ(70) downstream regulated genes including ftsZ, mazF, prfB, rpoS, seqA, turfB and ygjD. The treatment of PPNA06 on mono- or multiple MDR-GBN infected human gastric mucosal epithelial cells demonstrated the complete inhibition on bacterial growth and no influence on morphology and growth of human cells. Also, PPNA06 did not show the induction of antibiotic resistance as compared with classical antibiotics in GNB. These findings firstly demonstrate that rpoD is potential target for developing antisense antibiotics, and indicate that peptide conjugates of anti-rpoD PNA are active against GNBs in vitro and in vivo. Our results offer a feasible strategy for treating MDR-GNB infections.

Novel Anion Liposome-Encapsulated Antisense Oligonucleotide Restores Susceptibility of Methicillin-Resistant Staphylococcus aureus and Rescues Mice from Lethal Sepsis by Targeting mecA

ABSTRACT β-Lactam resistance in methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is caused by the production of an additional low-affinity penicillin-binding protein 2a, which is encoded by the mecA gene. The disruption of mecA may inhibit mecA expression and thereafter lead to the restoration of MRSA susceptibility to β-lactams. In this study, we developed a novel anionic liposome for encapsulating and delivering the complexes of a specific anti-mecA phosphorothioate oligodeoxynucleotide (PS-ODN833) and polycation polyethylenimine (PEI). The efficiencies of liposome encapsulation of the complexes were around 79.7% ± 2.7%. The liposomes showed sustained release of PS-ODN833 at 37°C but very low levels of release at 4°C and room temperature. The addition of the encapsulated anti-mecA PS-ODN833-PEI complex to cultures of MRSA strains caused 45, 76, 82, and 93% reductions in mecA expression, accompanied by the inhibition of MRSA growth on Mueller-Hinton agar containing oxacillin (6 μg/ml) in a concentration-dependent manner. The encapsulated-PS-ODN833 treatment also reduced the MICs of five of the most commonly used antibiotics for MRSA clinical isolates to values within the sensitivity range and rescued mice from MRSA-caused septic death by downregulating mecA. The survival rates of septic mice increased from 0% for the control group to 53% for the PS-ODN833-treated group. The results were associated with reductions of bacterial titers in the blood of surviving mice. The findings of the present study indicate that an antisense oligodeoxynucleotide targeted to mecA can significantly restore the susceptibility of MRSA to existing β-lactam antibiotics, providing an apparently novel strategy for treating MRSA infections.

Arrhythmogenic Effect of Sympathetic Histamine in Mouse Hearts Subjected to Acute Ischemia

The role of histamine as a newly recognized sympathetic neurotransmitter has been presented previously, and its postsynaptic effects greatly depended on the activities of sympathetic nerves. Cardiac sympathetic nerves become overactivated under acute myocardial ischemic conditions and release neurotransmitters in large amounts, inducing ventricular arrhythmia. Therefore, it is proposed that cardiac sympathetic histamine, in addition to norepinephrine, may have a significant arrhythmogenic effect. To test this hypothesis, we observed the release of cardiac sympathetic histamine and associated ventricular arrhythmogenesis that was induced by acute ischemia in isolated mouse hearts. Mast cell-deficient mice (MCDM) and histidine decarboxylase knockout (HDC−/−) mice were used to exclude the potential involvement of mast cells. Electrical field stimulation and acute ischemia-reperfusion evoked chemical sympathectomy-sensitive histamine release from the hearts of both MCDM and wild-type (WT) mice but not from HDC−/− mice. The release of histamine from the hearts of MCDM and WT mice was associated with the development of acute ischemia-induced ventricular tachycardia and ventricular fibrillation. The incidence and duration of induced ventricular arrhythmias were found to decrease in the presence of the selective histamine H2 receptor antagonist famotidine. Additionally, the released histamine facilitated the arrhythmogenic effect of simultaneously released norepinephrine. We conclude that, under acute ischemic conditions, cardiac sympathetic histamine released by overactive sympathetic nerve terminals plays a certain arrhythmogenic role via H2 receptors. These findings provided novel insight into the pathophysiological roles of sympathetic histamine, which may be a new therapeutic target for acute ischemia-induced arrhythmias.

Targeting RNA Polymerase Primary σ70 as a Therapeutic Strategy against Methicillin-Resistant Staphylococcus aureus by Antisense Peptide Nucleic Acid

Background Methicillin-resistant Staphylococcus aureus (MRSA) causes threatening infection-related mortality worldwide. Currently, spread of multi-drug resistance (MDR) MRSA limits therapeutic options and requires new approaches to “druggable” target discovery, as well as development of novel MRSA-active antibiotics. RNA polymerase primary σ70 (encoded by gene rpoD) is a highly conserved prokaryotic factor essential for transcription initiation in exponentially growing cells of diverse S. aureus, implying potential for antisense inhibition. Methodology/Principal Findings By synthesizing a serial of cell penetrating peptide conjugated peptide nucleic acids (PPNAs) based on software predicted parameters and further design optimization, we identified a target sequence (234 to 243 nt) within rpoD mRNA conserved region 3.0 being more sensitive to antisense inhibition. A (KFF)3K peptide conjugated 10-mer complementary PNA (PPNA2332) was developed for potent micromolar-range growth inhibitory effects against four pathogenic S. aureus strains with different resistance phenotypes, including clinical vancomycin-intermediate resistance S. aureus and MDR-MRSA isolates. PPNA2332 showed bacteriocidal antisense effect at 3.2 fold of MIC value against MRSA/VISA Mu50, and its sequence specificity was demonstrated in that PPNA with scrambled PNA sequence (Scr PPNA2332) exhibited no growth inhibitory effect at higher concentrations. Also, PPNA2332 specifically interferes with rpoD mRNA, inhibiting translation of its protein product σ70 in a concentration-dependent manner. Full decay of mRNA and suppressed expression of σ70 were observed for 40 µM or 12.5 µM PPNA2332 treatment, respectively, but not for 40 µM Scr PPNA2332 treatment in pure culture of MRSA/VISA Mu50 strain. PPNA2332 (≥1 µM) essentially cleared lethal MRSA/VISA Mu50 infection in epithelial cell cultures, and eliminated viable bacterial cells in a time- and concentration- dependent manner, without showing any apparent toxicity at 10 µM. Conclusions The present result suggested that RNAP primary σ70 is a very promising candidate target for developing novel antisense antibiotic to treat severe MRSA infections.

Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through β-Catenin

Summary Glucagon-like peptide 1 (GLP-1) plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4) promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These functions were accompanied by an increase in osteoblast number and serum bone formation markers, while the adipocyte number was decreased. Furthermore, GLP-1 receptor was detected in bone marrow stromal cells (BMSCs), but not in osteoblast. Activation of GLP-1 receptor by Ex-4 promoted the osteogenic differentiation and inhibited BMSC adipogenic differentiation through regulating PKA/β-catenin and PKA/PI3K/AKT/GSK3β signaling. These findings reveal that GLP-1 receptor regulates BMSC osteogenic differentiation and provide a molecular basis for therapeutic potential of GLP-1 against osteoporosis.

RESTORATION OF ANTIBIOTIC SUSCEPTIBILITY IN METHICILLIN‐RESISTANT STAPHYLOCOCCUS AUREUS BY TARGETING MECR1 WITH A PHOSPHOROTHIOATE DEOXYRIBOZYME

1 Methicillin resistance in Staphylococcus aureus is mediated by the mecA gene. The mecA gene encodes a penicillin‐binding protein (PBP2a) possessing low β‐lactam affinity. Transcription of mecA is regulated by a signal transduction system consisting of the sensor/transducer MecR1. Disruption of the MecR1 regulatory pathway may inhibit mecA expression and restore methicillin‐resistant Staphylococcus aureus (MRSA) susceptibility to β‐lactams. 2 In the present study, a phosphorothioate deoxyribozyme (named PS‐DRz147) specifically targeting MecR1 mRNA was designed, synthesised and introduced into the MRSA strain WHO‐2. 3 The expression of mecR1 and mecA was inhibited by PS‐DRz147 in a concentration‐dependent manner. Consequently, the susceptibility of WHO‐2 colonies to the antibiotic oxacillin was restored. 4 The results of the present study indicate that blockade of the MecR1–MecI–MecA signalling pathway with an mecR1‐targeted DNAzyme can restore the susceptibility of MRSA to existing β‐lactam antibiotics.

Synthesis of Biscoumarin and Dihydropyran Derivatives and Evaluation of Their Antibacterial Activity

In an attempt to find a new class antibacterial agents, a series of biscoumarins (1–4) and dihydropyrans (5–13) were successfully prepared. The molecular structures of four representative compounds, that is, 4, 5, 8 and 12 were confirmed by single crystal X-ray diffraction study. These synthesized compounds were screened for their antibacterial activity in vitro against Staphylococcus aureus (S. aureus ATCC 29213), methicillin-resistant S. aureus (MRSA XJ 75302), vancomycin-intermediate S. aureus (Mu50 ATCC 700699), USA 300 (Los Angeles County clone, LAC), Staphylococcus epidermidis (S. epidermidis ATCC 14990), methicillin-resistant S. epidermidis (MRSE XJ 75284) and Escherichia coli (E. coli ATCC 25922). Additionally, there are two classical intramolecular O–H···O hydrogen bonds (HBs) in biscoumarins 1–4 and the corresponding HB energies were further performed with the density functional theory (DFT) [B3LYP/6-31G*] method.

Underlying Mechanism of In vivo and In vitro Activity of C-terminal–amidated Thanatin Against Clinical Isolates of Extended-Spectrum β-lactamase–Producing Escherichia coli

BACKGROUND Infections with extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) have developed resistance to current therapies. Therefore, the underlying mechanisms of in vivo and in vitro activity of C-terminal-amidated thanatin (A-thanatin) against clinical isolates of ESBL-EC were studied in an attempt to resolve this problem. METHODS A-thanatin was synthesized to determine its minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and kill curve for ESBL-EC. The hemolytic toxicity, stability, and resistance induction of A-thanatin were determined. ESBL-EC-infected mice were used to determine the in vivo activity of A-thanatin. Scanning and transmission electron microscopy and fluorescence microscopy were used to study the underlying mechanism of A-thanatin. RESULTS A-thanatin is highly effective against ESBL-EC in vitro, with MIC values ≤4 μg/mL. It has been confirmed that A-thanatin has little hemolysis and relative high stability in plasma. Excellent in vivo therapeutic effects were also observed in a septicemic animal model, with survival rates of 50.0%, 66.7%, and 91.7% in the low-dose, middle-dose, and high-dose groups, respectively. Membrane permeabilization may be a major biological action of A-thanatin. CONCLUSIONS Because the development of multidrug resistance limits the available therapeutic options, A-thanatin may provide a novel strategy for treating ESBL-EC infection and other infections due to multidrug-resistant bacteria.

Restoration of oxacillin susceptibility in methicillin-resistant Staphylococcus aureus by blocking the MecR1-mediated signaling pathway

Abstract The signal transducing integral membrane protein, MecR1 helps initiate the expression of the antibiotic-resistant gene mecA, which encodes the penicillin-binding protein 2a. MecA participates in the β-lactam resistance of methicillin-resistant Staphylococcus aureus (MRSA). Blocking the MecR1 regulatory pathway may be a novel strategy to combat MRSA. In this study, we introduced an antisense phosphothioate oligodeoxynucleotide (PS-ODN) targeting MecR1 mRNA into the MRSA strain WHO-2, which led to a significant reduction of both MecR1 and PBP2a mRNAs in a concentration-dependent manner. Consequently, the susceptibility of S. aureus WHO-2 to the β-lactam antibiotic oxacillin was restored significantly. Our results indicate that blocking the mecR1-mecI-mecA signaling pathway via an antisense approach might be a viable strategy to restore the susceptibility of MRSA to the existing β-lactam antibiotics.