Solid tumors grow within a complex microenvironment composed of diverse cell types such as fibroblasts, endothelial cells, mast cells, macrophages and immune cells that are attracted by tumor cell derived factors and embedded in an extracellular matrix.Molecular and cellular interactions between epithelial cells and cells surrounding the tumor stroma promote growth, invasion and spread of tumors.To delay or impede tumor growth, the tumor microenvironment (TME) is increasingly being explored as a potential therapeutic target for which novel strategies are developed.lt;/pgt;lt;pgt; This article reviews how key interactions between tumor cells and surrounding mesenchymal and immune cells in the TME can promote tumor progression and it highlights cellular and molecular elements that might represent novel therapeutic targets.Special emphasis is given on therapies targeted towards tumor-associated macrophages.As main class of drugs the bisphosphonates are covered with their properties to repolarize a pro-tumorigenic, immunosuppressive environment to a tumor growth inhibiting and immunocompetent microenvironment.Properties and advantages of liposome-encapsulated bisphosphonates as macrophage depleting or modulating agents as well as the latest developments towards clinical applications of compounds targeting cellular and molecular components of the TME are described and reviewed.

The treatment of septicemia caused by antibiotic-resistant bacteria is a great challenge in the clinic. Because traditional antibiotics inevitably induce bacterial resistance, which is responsible for many treatment failures, there is an urgent need to develop novel antibiotic drugs. Amino-terminated Poly(amidoamine) dendrimers (PAMAM-NH2) are reported to have antibacterial activities. However, previous studies focused on high generations of PAMAM-NH2, which have been found to exhibit high toxicities. The present study aimed to clarify whether low generations of PAMAM-NH2 could be used as novel antibacterial agents. We found that generation 2 (G2.0) PAMAM-NH2 showed significant antibacterial effects against antibiotic-sensitive and antibiotic-resistant strains but exhibited little toxicity to human gastric epithelial cells and did not induce antibiotic resistance in bacteria. Scanning and transmission electron microscopy analyses suggested that G2.0 PAMAM-NH2 might inhibit the growth of bacteria by destroying their cell membranes. The administration of G2.0 PAMAM-NH2 dose-dependently improved the animal survival rate of mice infected with extended-spectrum beta lactamase-producing Escherichia coli (ESBL-EC) and of animals infected with a combination of ESBL-EC and methicillin-resistant Staphylococcus aureus. A treatment regimen of 10 mg/kg of G2.0 PAMAM-NH2 starting 12 h before inoculation followed by 10 mg/kg at 0.5 h after inoculation rescued 100% of singly infected mice and 60% of multiply infected mice. The protective effects were associated with the reduction of the bacterial titers in the blood and with the morphological amelioration of infected tissues. These findings demonstrate that the G2.0 PAMAM-NH2 is a potential broad-spectrum and nonresistance-inducing antibiotic agent with relatively low toxicity.

In consideration of high production costs of new antimicrobial drugs, a more convenient and economical method for time–kill study is urgently required. In the present experiment, we attempted to demonstrate the feasibility of microplate method as an alternative measure of macrodilution method for time–kill study. Three conventional antibiotics (ciprofloxacin, ceftazidime, and levofloxacin) and two antimicrobial peptides [A-thanatin and K4-S4(1–16)a] were used to determine time–kill curves against Escherichia coli ATCC 25922 and Staphylococcus epidermidis ATCC 14990. Meanwhile, both methods were also performed with three antisense peptide nucleic acids (PNA3, PNA4, and PNA5) targeting ropD gene of Staphylococcus aureus ATCC 29213 and MRSA WHO-2. In order to study the correlation between the two methods, the growth inhibition rate of PNAs, antimicrobial peptides, and antibiotics for the tested strains were evaluated. A strong agreement between the results obtained from the two methods has been demonstrated. Although microplate method required longer incubation time for a significant result than macrodilution method, the former provides a more convenient, economical, and stable way to perform time–kill test for these agents. Thus, we concluded that microplate method was an available measure for time–kill study of new antimicrobial drugs.

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.

Recent years have witnessed several gram-negative bacteria (GNB) species and a few grampositive bacteria (especially the Staphylococcus aureus) posing overwhelming threats to the healthcare-associated infections as a series of frightening superbugs (Engel, 2010; Peleg & Hooper, 2010). It is primarily due to the fact that incidence of multidrug resistance (MDR) or pan-drug resistance (PDR) bacteria have been escalating in a manner of global dimension, frequent prevalence and alarming magnitude. The predominate resistance issues are those related to GNB species, including Enterobacteriaceae (Deshpande & et al, 2010), Klebsiella pneumonia, Pseudomonas aeruginosa and Acinetobacter baumannii. Theses circulating isolates have created big problems for treatment of nosocomial infection because they carry highly transmissible elements encoding multiple resistance genes, e.g. extended-spectrum betalactamases (ESBLs) that inactivates different classes of first-line antibiotics (Bush, 2010; Engel, 2010), metallo-beta lactamase that hydrolyzes penicillins, cephalosporins and carbapenems, efflux pumps that decrease bacterial transporting ability to almost all antibiotics and natural antimicrobial products (Pages & et al, 2010), and promoters that ensure the transcription of these genes.

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.

细菌的转录过程是一个由多种分子共同调控的复杂过程,其中RNA聚合酶(RNA polymerase,RNAP)是催化转录合成RNA的重要酶.作为RNAP中一个独立的亚单位,σ因子(sigma factor)在转录起始过程中起着至关重要的作用.最近的研究表明σ因子参与了转录起始的各个过程,包括启动子的定位、启动子的解链、起始RNA合成、脱离启动子等过程.由于其在细菌转录过程中的重要作用,σ因子正在成为抗菌药物研究的新靶点.本文对σ因子的结构、分类、功能以及以它为中心的调控网络的研究进行综述.

The antimicrobial peptide is a class of small molecule peptide derived from a variety of creatures, able to kill the pathogens efficiently and has the potential of broad-spectrum activity, rapid and strong bactericidal activity, low propensity for resistance development, and many other advantages. As a new generation of anti-infective drug candidates, the mechanism of antimicrobial peptides has not yet entirely clear, but now there are two views have been widely recognized that the destruction of permeation through the membrane structural integrity and through the different intracellular target to affect bacterium growth and metabolic balance. In this paper, the physical and chemical properties, secondary structure, mechanism, and the relationship between the latter two to make a conclusion, in order to better understand the structure-activity relationships and to provide a theoretical basis for rational design of antimicrobial peptides.

Fluoroquinolone-resistant Escherichia coli (FREC) is one of the leading causes of Gram-negative bacterial infections short of effective antibiotics, thus necessitating development of novel antibacterial agents such as antisense resistance inhibitors. Aiming to restore susceptibility of FREC to fluoroquinolones by antisense inhibition of essential resistance mechanism, we designed and synthesized anion liposome encapsulated phosphorothioate oligodeoxynucleotide 831 (PS-ODN831) targeting gene acrB , which encodes the AcrAB–TolC efflux pump responsible for decreasing intercellular antibiotic concentrations. In all encapsulated PS-ODN831-treated groups, the MICs of ciprofloxacin and levofloxacin to FREC were reduced at different degrees, therefore inhibiting growth of FREC in a concentration-dependent manner. Reversion of their bactericidal effects was the result of specific and potent inhibition of acrB mRNA and the activity of efflux pump of AcrAB–TolC in FREC strains by liposome-encapsulated PS-ODN831. The study indicated that antisense targeting of AcrAB–TolC efflux pump system may be a feasible and potential strategy to treat FREC infections.

The progress of transcription is synthesized by complex molecules, among which DNA-dependent RNA polymerase (RNAP) is the central enzyme. The prokaryotic RNAP is a large protein composed of core subunits (α2, β and β') and a σ factor that is required for specific recognition of the promoter site and the initiation of transcription. Despite its ubiquity, structural and functional similarities, bacterial RNAPs do not share extensive sequence homology with eukaryotic RNAPs. Bacterial RNAP an attractive target for the development of anti-bacterial drugs as its inactivation would lead to bacterial cell death. This review will present the state of knowledge on the assembly and function of RNAP subunits in bacteria with special focus on insights provided by structural analysis of a key component σ factor. Thorough retrospection has been provided for better understanding of progress and problems in targeting RNAP by traditional chemical compounds. Recent progress using innovative strategies including structural biology and phage based screening, especially the antisense technology, has shed light on developing the first set of macro-molecule RNAP inhibitors. In particular, exploration on targeting RNAP σ70 for realization of broad spectrum antisense bactericidal effect in gram negative bacteria presents the first successful example of PNA-peptide conjugate showing attractive potential as conventional broad-spectrum antibiotics, in which possible way the antisense antibiotics might develop into to meet the range and type of usage in future health care.

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.

To find potential enhancers for facilitating the buccal delivery of insulin, a TR146 cell-culture model of buccal epithelium, cultured on commercially available insert plates, was used to evaluate the permeability-enhancing effects of several traditional and new types of chemical enhancers, including N-acetyl-L-cysteine (NAC), sodium deoxycholate (SDC), sodium nitroprusside (SNP), reduced glutathione (GSH), glutamine (Gln), chitosan (CS), L-arginine (Arg), 1-dodecylazacycloheptan-2-one (Azone), and soybean lecithin (SPC) (50 and 10 μg/mL respectively). Permeability studies were performed to determine the enhancing effects of these compounds on insulin permeation across the cell-culture model. The enhancing effects of the enhancers were assessed by calculating the apparent permeability coefficients and enhancement ratio. Cytotoxicity of the permeation enhancers at different concentrations was investigated by using the methylthiazolydiphenyl-tetrazolium bromide (MTT) assay. Results showed that 50 μg/mL of NAC, SDC, GSH, CS, Arg, Azone, SPC, SNP, and 10 μg/mL of SNP had a significant enhancing effect on promoting the transport of insulin across the TR146 cell model. MTT assays showed that 50 μg/mL of Gln, Azone, SDC, SNP, Arg, 10 μg/mL SDC, and Arg had obvious toxic effects on TR146 cells. Therefore, NAC, GSH, CS, SPC, and SNP appear to be safe, effective permeability enhancers that promote the transport of insulin across the TR146 cell-culture model of buccal epithelium and may be potential enhancers for buccal delivery of insulin with both low toxicity and high efficiency.

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) is caused by the production of low-affinity penicillin-binding protein 2a and β-lactamases, which are encoded by mecA and blaZ, respectively.Expressions of the two key genes are mutually regulated by MecI and BlaI.The aim of this study was to design specific anti-mecR1 and anti-blaR1 deoxyribozymes and identify the restoration of susceptibility in MRSA isolates with mecI or blaI or no deletions by interfering with the mutual regulation of mecA and blaZ.Material and methods: Specific deoxyribozymes were designed by using the program RNA structure 4.6.RNA substrates were obtained by transcription in vitro and used to assess the target cleavage of DNAzymes.Transcription of mecR1-mecA and blaR1-blaZ was analysed by real time RT-PCR.The susceptibility of MRSA was tested.Results: Specific deoxyribozymes showed efficient catalytic activity to each own substrate mecR1 or blaR1 in vitro and caused the reduction of mecR1 and blaR1 transcription in vivo.Furthermore, simultaneous administration of two DNAzymes to knockdown mecR1 and blaR1 resulted in increased susceptibility of all MRSA strains tested in this study.Conclusions: These results demonstrated that combined use of the two specific phosphorothioate deoxyribozymes could be a viable and promising strategy to restore the susceptibility of almost all MRSA clinical isolates.

The purpose of this study was to exploit the potential of Primer Premier 5. 0 and RNA structure 4. 6 in the design and selection of effective 10-23 deoxyribozyme ( DRz) targeting resistance gene mecR1. Five designed and synthesized anti-mecR1 10-23 DRz sequences were introduced into the MRSA strain by electro transformation in vivo. Transcription of mecR1 was analyzed by real-time quantitative PCR. The inhibitory effects of DRzs on the bacterial growth were evaluated based on the plate cloning formation. It was found that the five anti-mecR1 10-23 DRz sequences significantly lowered the transcription of mecR1 and inhibited the growth of clinical drug-resistant MRSA080309,with DRz6 having the most significant inhibitory effect. Therefore,the combination of the two computer softwares is an economical,practical and effective approach in the design of anti-mecR1 DRz,and could greatly reduce the screening time of antisense drugs.

Multidrug-resistant Pseudomonas aeruginosa (MDR-PA) is one of the leading Gram-negative organisms associated with nosocomial infections. The increasing frequency of MDR-PA has represented a huge challenge in conventional antibacterial therapy. The loss of effectiveness of commonly used antibiotics calls for the immediate need to develop an alternative strategy for combating MDR-PA infections. The multiantibiotic resistance of MDR-PA is largely attributable to the production of multidrug efflux pumps, MexAB-OprM. OprM forms the antibiotic-ejecting duct and plays a crucial role in exporting incoming chemotherapeutic agents across the membranes. Disruption of the OprM expression may inhibit the function of multidrug efflux pumps and lead to restoration of MDR-PA susceptibility to antibiotics. In this study, we developed a novel anion liposome for encapsulating and delivering specific anti-oprM phosphorothioate oligodeoxynucleotide (PS-ODN617) and polycation polyethylenimine (PEI) complexes. The additions of the encapsulated anti-oprM PS-ODN617/PEI to MDR-PA isolates caused a significant reduction of oprM expression and inhibition of MDR-PA growth in the presence of piperacillin in a concentration-dependent manner. The encapsulated PS-ODN617 treatment also reduced minimal inhibitory concentrations of five most commonly used antibiotics to the sensitive margin values on MDR-PA clinical isolates, respectively. The results of present study firstly indicate that PS-ODN targeted to oprM can significantly restore the susceptibility of MDR-PA to existing antibiotics, which appears to be a novel strategy for treating MDR-PA infections.

Antimicrobial peptides are cationic amphiphilic polypeptides produced by almost all species of lives as an important immune defense component,and have the potential of broad-spectrum activity,rapid and strong bactericidal activity,low propensity for resistance development,and many other advantages.With the multi-drug resistant clinical infection becoming increasingly heavier,these peptides have aroused extensive attention as a kind of great potential for development of new anti-infective agents.However,their high production cost,toxic side effects and lack of pharmacokinetics information in vivo have seriously hampered the research and application of antimicrobial peptides.In response to these bottlenecks,a large number of studies carried out at home and abroad,have achieved remarkable results.In this paper,the characteristics and mechanisms of action,research status,application prospects and problems in the field of medicine are reviewed.

OBJECTIVE:To screen formulation of Insulin cream and to investigate the effects of insulin cream on wound healing in rabbits.METHODS:The best formulation of insulin cream was selected from 8 kinds of formulations with appearance and stability as index.Rabbit trauma model were induced and divided into matrix group,blank group,Insulin cream groups(high-dose,medium-dose and low-dose).Wound healing rate and healing time were compared among those groups.RESULTS:The fifth formulation possessed good appearance and stability,which included albolene,cetanol,glycerin monostearate,glycerol,etc.Compared with matrix group,the wound healing rates were obviously increased at different time points in Insulin cream groups(P0.05),and the wound healing period was significantly shortened(P0.05).CONCLUSION:The quality of optimized insulin scream conform with the standard.Local application of Insulin cream improves wound healing in rabbits.

The nightmare of multi-drug resistant bacteria will still haunt if no panacea is ever found. Efforts on seeking desirable natural products with bactericidal property and screening chemically modified derivatives of traditional antibiotics have lagged behind the emergence of new multi-drug resistant bacteria. The concept of using antisense antibiotics, now as revolutionary as is on threshold has experienced ups and downs in the past decade. In the past five years, however, significant technology advances in the fields of microbial genomics, structural modification of oligonucleotides and efficient delivery system have led to fundamental progress in the research and in vivo application of this paradigm. The wealthy information provided in the microbial genomics era has allowed the identification and/or validation of a number of essential genes that may serve as possible targets for antisense inhibition; antisense oligodeoxynucleotides (ODNs) based on the 3rd generation of modified structures, e.g., peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs) have shown great potency in gene expression inhibition in a sequence-specific and dosedependent manner at low micromolar concentrations; and cell penetrating peptide mediated delivery system has enabled the effective display of intracellular antisense inhibition of targeted genes both in vitro and in vivo. The new methods show promise in the discovery of novel gene-specific antisense antibiotics that will be useful in the future battle against drug-resistant bacterial infections. This review describes this promising paradigm, the targets that have been identified and the recent technologies on which it is delivered.

Carbohydrates are proved to be involved in recognition processes, including adhesion between cells, adhesion of cells to the extracellular matrix, and specific recognition of cells by one another. Galectin-3 is a nonenzymatic carbohydrate-binding protein present in mammals, whose conserved carbohydrate-recognition domain preferentially binds to specific carbohydrate structures and plays an important role in biological and biochemical reactions. Substances developed with the specific structure of beta-galactose moieties or their analogues, including chemically modified carbohydrates, functional peptides and modified natural polysaccharide, have been evaluated as potent therapeutic ligands for galectin-3 and showed at different level their ability to interfere with carbohydrate-protein interactions and therefore, inhibit the cell-cell recognition and adhesion processes, which play an important role in tumor growth, progression and metastasis.