Abstract Background Outbreaks of infection due to multidrug-resistant (MDR) bacteria, especially Gram-negative bacteria, have become a global health issue in both hospitals and communities. Antisense oligonucleotides (ASOs) based therapeutics hold a great promise for treating infections caused by MDR bacteria. However, ASOs therapeutics are strangled because of its low cell penetration efficiency caused by the high molecular weight and hydrophilicity. Results Here, we designed a series of dendritic poly-peptides (DPP1 to DPP12) to encapsulate ASOs to form DSPE-mPEG2000 decorated ASOs/DPP nanoparticles (DP-AD1 to DP-AD12) and observed that amphipathic DP-AD2, 3, 7 or 8 with a positive charge ≥ 8 showed great efficiency to deliver ASOs into bacteria, but only the two histidine residues contained DP-AD7 and DP-AD8 significantly inhibited the bacterial growth and the targeted gene expression of tested bacteria in vitro. DP-AD7anti-acpP remarkably increased the survival rate of septic mice infected by ESBLs-E. coli, exhibiting strong antibacterial effects in vivo. Conclusions For the first time, we designed DPP as a potent carrier to deliver ASOs for combating MDR bacteria and demonstrated the essential features, namely, amphipathicity, 8–10 positive charges, and 2 histidine residues, that are required for efficient DPP based delivery, and provide a novel approach for the development and research of the antisense antibacterial strategy. Graphical Abstract

Bacterial cell division is a highly controlled process regulated accurately by a diverse array of proteins spatially and temporally working together. Among these proteins, FtsZ is recognized as a cytoskeleton protein because it can assemble into a ring-like structure called Z-ring at midcell. Z-ring recruits downstream proteins, thus forming a multiprotein complex termed the divisome. When the Z-ring scaffold is established and the divisome matures, peptidoglycan (PG) biosynthesis and chromosome segregation are triggered. In this review, we focus on multiple interactions between FtsZ and its accessory proteins in bacterial cell cytokinesis, including FtsZ localization, Z-ring formation and stabilization, PG biosynthesis, and chromosome segregation. Understanding the interactions among these proteins may help discover superior targets on treating bacterial infectious diseases.

BACKGROUND:Antisense oligonucleotides (ASOs) based technology is considered a potential strategy against antibiotic-resistant bacteria; however, a major obstacle to the application of ASOs is how to deliver them into bacteria effectively. DNA tetrahedra (Td) is an emerging carrier for delivering ASOs into eukaryotes, but there is limited information about Td used for bacteria. In this research, we investigated the uptake features of Td and the impact of linkage modes between ASOs and Td on gene-inhibition efficiency in bacteria.RESULTS:Td was more likely to adhere to bacterial membranes, with moderate ability to penetrate into the bacteria. Strikingly, Td could penetrate into bacteria more effectively with the help of Lipofectamine 2000 (LP2000) at a 0.125 μL/μg ratio to Td, but the same concentration of LP2000 had no apparent effect on linear DNA. Furthermore, linkage modes between ASOs and Td influenced gene-knockdown efficiency. Looped structure of ASOs linked to one side of the Td exhibited better gene-knockdown efficiency than the overhung structure.CONCLUSIONS:This study established an effective antisense delivery system based on loop-armed Td, which opens opportunities for developing antisense antibiotics.

Multidrug resistance poses a severe threat to public health and urgently requires new solutions. The natural product chelerythrine (CHE) is a benzophenanthridine alkaloid with antimicrobial potential. In this study, CHE was effective against seven gram-positive bacterial strains, and the minimum inhibitory concentrations (MICs) ranged from 2 to 4 μg/mL. By contrast, CHE showed inferior antibacterial activities against 11 gram-negative strains, and the MICs varied from 16 to 256 μg/mL. We also determined the synergistic/additive effects of combining CHE with nine currently used antibiotics. CHE restored the antibacterial efficacy of the antibiotics against methicillin-resistant Staphylococcus aureus and extended-spectrum β-lactamases producing Escherichia coli. This study suggests that the combination of CHE with conventional antibiotics may be a promising strategy to combat infections caused by multidrug-resistant organisms.

The emergence and rapid spread of multidrug resistance in bacteria have led to the urgent need for novel antibacterial agents. Membrane permeabilization is the mechanism for many antibacterial molecules that are being developed against gram-negative bacteria. Thus, to determine the efficacy of a potential antibacterial molecule, it is important to assess the change in bacterial membrane permeability after treatment. This study describes the protocol for the assays of outer and inner membrane permeability using the fluorescent probes N-phenyl-1-naphthylamine and propidium iodide. Compared with other experiments, such as electron microscopy and the assay of minimal bactericidal concentration, this methodology provides a simpler, faster, and cost-effective way of estimating the membrane-permeabilizing effect and bactericidal efficacy of antibacterial molecules. This study presents an optimized protocol with respect to the classical protocols by incubating bacteria with antibacterial molecules in the culture condition identical to that of antibacterial assays and then detecting the signal of the fluorescent probe in the buffer without broth and antibacterial molecules. This protocol avoids the effect of nutrient deficiency on the physiological status of bacteria and the interference of antibacterial molecules towards the fluorescent probe. Thus, this method can effectively and precisely evaluate the membrane permeability and match the results obtained from other antibacterial assays, such as minimum inhibitory concentration and time-kill curve assays.

Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonist exendin-4 (Ex-4), a drug that has been used in the clinical treatment of type 2 diabetes mellitus, also confers a neuroprotective effect against stroke. Although GLP-1 analogs were reported to induce sustained insulin secretion and glucose tolerance improved after cessation of treatment, no study has revealed whether Ex-4 exerts sustained neuroprotection against stroke and the underlying mechanism after treatment cessation. In this study, mice were pretreated with Ex-4 for 7 days, and middle cerebral artery occlusion (MCAO) was performed on different days after cessation of Ex-4 treatment. Ex-4 ameliorated neurological dysfunction and reduced the infarct volume induced by MCAO. These protective effects lasted for 6 days after the cessation of Ex-4 treatment and were associated with sustained upregulation of PI3K, AKT, mTOR, and HIF-1α levels, as well as HIF-1α downstream genes. Knockdown of GLP-1R or HIF-1α in the brain by short hairpin RNA abolished Ex-4 treatment-mediated neuroprotection. In normal mice, Ex-4 treatment led to instant upregulation of p-PI3K, p-AKT, p-mTOR, and HIF-1α expression levels, which quickly returned to normal after cessation of Ex-4 treatment, while the expression levels of insulin growth factor-1 receptor (IGF-1R) remained high for 6 days after Ex-4 cessation. Additionally, Ex-4 did not directly induce IGF-1 production, which was only induced by MCAO. Ex-4 induces extended cerebral ischemic tolerance. This neuroprotective effect is associated with activation of GLP-1R and upregulation of IGF-1R in the brain, and the latter then activates the PI3K/AKT/mTOR/HIF-1 signaling pathway via binding to IGF-1 secreted from the ischemic brain.

Staphylococcus aureus infection is difficult to eradicate because of biofilm formation and antibiotic resistance. The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infection necessitates the development of a new agent against bacterial biofilms. We report a new coumarin compound, termed DCH, that effectively combats MRSA in vitro and in vivo and exhibits potent antibiofilm activity without detectable resistance. Cellular proteome analysis suggests that the molecular mechanism of action of DCH involves the arginine catabolic pathway. Using molecular docking and binding affinity assays of DCH, and comparison of the properties of wild-type and ArgR-deficient MRSA strains, we demonstrate that the arginine repressor ArgR, an essential regulator of the arginine catabolic pathway, is the target of DCH. These findings indicate that DCH is a promising lead compound and validate bacterial ArgR as a potential target in the development of new drugs against MRSA biofilms.

Proadrenomedullin N-terminal 20 peptide (PAMP) is elevated in sepsis, but the function and possible mechanism of PAMP in bacterial infection is elusive. This study is aim to evaluate the role of PAMP in the interaction between the Enterohemorrhagic E. coli (EHEC) and the host barrier. Our results showed that PAMP alleviated the EHEC-induced disruption of goblet cells and mucosal damage in the intestine, increased the expression of occludin in the colon of EHEC-infected mice, and reduced the proinflammatory cytokines level in serum significantly compared with the control group. Meanwhile, lipopolysaccharide (LPS) stimulation could dose-dependently induce the expression of preproADM, the precursor of PAMP, in human intestinal epithelial cell (HIEC) and human umbilical vein endothelial cell (HUVEC). In addition, PAMP inhibited the growth of EHEC O157:H7 and destroyed the inner and outer membrane. At low concentration, PAMP attenuated the EHEC virulence genes including hlyA and eaeA, which was also confirmed from reduced hemolysis to red cells and adhesion to HIEC. These results indicated that EHEC infection would modulate the expression of PAMP in intestinal epithelium or vascular endothelium, and in turn exerted a protective effect in EHEC induced infection by rupturing the bacterial cell membrane and attenuating the bacterial virulence.

Acinetobacter baumannii (A. baumannii) is an important opportunistic pathogen causing serious nosocomial infections, which is considered as the most threatening Gram-negative bacteria (GNB). Outer membrane protein A (OmpA), a major component of outer membrane proteins (OMPs) in GNB, is a key virulence factor which mediates bacterial biofilm formation, eukaryotic cell infection, antibiotic resistance and immunomodulation. The characteristics of OmpA in Escherichia coli (E. coli) have been extensively studied since 1974, but only in recent years researchers started to clarify the functions of OmpA in A. baumannii. In this review, we summarized the structure and functions of OmpA in A. baumannii (AbOmpA), collected novel therapeutic strategies against it for treating A. baumannii infection, and emphasized the feasibility of using AbOmpA as a potential therapeutic target.

目的:观察RNAⅢ抑制肽衍生物RIP1183对SD大鼠、Beagle犬的急性毒性反应和对豚鼠过敏反应.方法:SD大鼠和Beagle犬随机分为RIP1183给药组和对照组.SD大鼠尾静脉注射RIP1183,单次剂量为50 mg·kg-1,间隔4～5 h,连续2次,总剂量为100 mg·kg-1,对照组给予等体积的0.9％氯化钠注射液,给药后连续14 d观察RIP1183对大鼠的毒性作用.Beagle犬静脉注射RIP1183,最大给药剂量为50 mg·kg-1,对照组给予等体积的0.9％氯化钠注射液,给药后连续14 d观察RIP1183对Beagle犬的体质量、体温、摄食量、血清生化指标、血液学指标、眼科检查和心电图的影响.采用豚鼠全身主动过敏实验,Hartley豚鼠随机分为阴性对照组(0.9％氯化钠注射液)、阳性对照组(牛血清白蛋白V)、RIP1183低(9.3 mg·kg-1)、高(18.6 mg·kg-1)剂量组,每组6只.动物致敏腹腔注射给药3次,激发静脉注射给药2次,激发给药后观察动物是否出现过敏反应.结果:给予RIP1183后,SD大鼠外观、行为活动、饮食等均未见明显异常,体质量增长正常,结束时全部存活,大体解剖未见肉眼可见变化.Beagle犬给药后,其体质量、体温和摄食量与对照组比较,差异无统计学意义(P>0.05),其他各项指标均无异常.阳性对照组豚鼠在激发后出现过敏症状,RIP1183各剂量组豚鼠激发后未见明显异常和过敏反应症状.结论:SD大鼠和Beagle犬分别静脉给予100 mg·kg-1和50 mg·kg-1 RIP1183后均无异常反应,亦无死亡;豚鼠的RIP1183过敏反应为阴性.

Background: The development of bacterial biofilm can cause severe chronic infections and antibiotic resistance. Therefore, it poses a significant threat to public health. Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) are two major pathogens that can cause biofilm-associated infections, which leads to the urgent necessity of developing new agents with biofilm-forming inhibitory ability. Methods: A series of pyran derivatives were synthesized and characterized, and their in vitro anti-biofilm activity against S. aureus and P. aeruginosa were measured by minimal biofilm inhibitory concentration assay and FITC dye staining. The in vivo antibiofilm therapeutical effects were evaluated in S. aureus induced tissue cage infection mice model and P. aeruginosa induced urinary tract catheter infection rat model. Results: Several pyran derivatives showed the in vitro anti-biofilm activity against S. aureus and P. aeruginosa, and the activity of these compounds was not mediated through the accessory gene regulator (agr) quorum sensing system of S. aureus. One of these pyran derivatives, namely 2-amino-4-(2,6-dichlorophenyl)-3-cyano-5-oxo-4H,5H-pyrano[3,2c]chromene, exhibited significant inhibitory biofilm-formation activity in S. aureus tissue cage infection mice model and in the P. aeruginosa-infected urinary tract catheters of experimental rats. Conclusions: The data indicated that this pyran derivative is a possible lead compound that can be used for the development of novel anti-biofilm agents against S. aureus and P. aeruginosa infection. (C) 2019 The Author(s). Published by Elsevier Ltd on behalf of King Saud Bin Abdulaziz University for Health Sciences.

Stroke is the second most frequent cause of mortality, resulting in a huge societal burden worldwide. Timely reperfusion is the most effective therapy; however, it is difficult to prevent ischemia/reperfusion (I/R) injury. In traditional Chinese medicine, hydroxysafflor yellow A (HSYA) has been widely used for the treatment of cerebrovascular disease and as a protective therapy against I/R injury. Evidence has demonstrated that HSYA could reduce the levels of reactive oxygen species and suppress cellular apoptosis; however, whether HSYA alters the metabolic profile as its underlying mechanism for neuroprotection remains unknown. In the present study, using a metabolomic screening, phenylalanine was identified to significantly increase in an experimental model of mouse cerebral I/R injury. Notably, western blotting and qPCR analysis were conducted to test the expression level of apoptosis-associated factors, and HSYA was identified to be able to protect neuronal cells by reducing phenylalanine level associated with I/R injury. Additionally, these findings were confirmed in primary mouse neurons and PC12 cells exposed to oxygen and glucose deprivation/reoxygenation (OGD/R) stress. Of note, HSYA was observed to regulate the mRNA expression of key metabolic enzymes, phenylalanine hydroxylase, tyrosine aminotransferase and aspartate aminotransferase, which are responsible for phenylalanine metabolism. Furthermore, by performing mitochondrial labeling and JC-1 fluorescence assay, HSYA was identified to promote mitochondrial function and biogenesis suppressed by OGD/R. The findings of the present study demonstrated that I/R injury could increase the levels of phenylalanine, and HSYA may inhibit phenylalanine synthesis to enhance mitochondrial function and biogenesis for neuroprotection. The present study proposed a novel metabolite biomarker for cerebral I/R injury and the evaluated the efficacy of HSYA as a potential therapeutic treatment I/R injury.

BACKGROUND:Carbapenem-resistant Acinetobacter baumannii (A. baumannii) was on the top of the list of the most threatening bacteria published by the WHO in 2017. Antisense oligonucleotides (ASOs) based therapy is a promising strategy for combating Multi-Drug Resistant (MDR) bacteria because of its high specificity, easy design and lower induction of resistance, but poor cellular uptake by bacteria has restricted the further utilization of this therapy.METHODS:Here, we used CADY, a secondary amphipathic peptide of 20 residues that could successfully carry siRNA into mammalian cells, to prepare CADY/ASOs nanoparticles (CADY-NPs) targeting acpP (encoding acyl carrier protein), and evaluated the uptake features, the inhibitory effects of CADY-NPs on gene expression and the growth of MDR-A. baumannii.RESULTS:We found that CADY-NPs could be quickly internalized by drug-sensitive and MDR-A. baumannii in an energy independent manner, which could be restrained by chlorpromazine (an inhibitor of clathrin mediated endocytosis) significantly. In addition, CADY-NPs targeting acpP concentrationdependently retarded the growth of MDR-A. baumannii, which was associated with the decreased expression of targeted genes in A. baumannii.CONCLUSION:In conclusion, our research is the first to demonstrate that CADY can deliver ASOs into bacteria and provide a novel strategy for the treatment of MDR-A. baumannii.

实验教学是医学生药理学教学过程中的一个重要环节,也是提升本科生创新思维的重要方式和途径.为了有效提升医学生创新能力,我们教研室经过多年的摸索,从优化药理学实验课程体系、增加自主性实验设计、开展探究式课外科研活动和实施多因素考核方案这四个方面对药理学实验课程体系进行了整体优化.经过多年实践表明,优化后的实验教学模式取得了显著成效.它给学生提供了广泛的自主学习空间,充分调动了学生的自主学习兴趣,营造出积极上进的学习氛围;加强了学生独立思考的意识和创新能力,提高了学生分析、解决问题的实践能力,使学生药理学知识结构系统化,其综合能力得到了全面协调的发展.

New Delhi metallo-β-lactamase-1 (NDM-1) is the most prevalent type of metallo-β-lactamase and hydrolyzes almost all clinically used β-lactam antibiotics. Here we show that the antimicrobial peptide thanatin disrupts the outer membrane of NDM-1-producing bacteria by competitively displacing divalent cations on the outer membrane and inducing the release of lipopolysaccharides. In addition, thanatin inhibits the enzymatic activity of NDM-1 by displacing zinc ions from the active site, and reverses carbapenem resistance in NDM-1-producing bacteria in vitro and in vivo. Thus, thanatin's dual mechanism of action may be useful for combating infections caused by NDM-1-producing pathogens.

目的 考察新型细菌群体抑制剂RNAⅢ抑制肽衍生物RIP1183对中枢神经系统、心血管系统和呼吸系统的影响.方法 将ICR种小鼠48只,随机分为对照组和RIP1183低、中、高剂量组(4、12、36mg/kg),观察iv RIP1183后,对小鼠自主活动次数、协调运动能力和阈下巴比妥钠协同作用的影响.将比格犬随机分组同前,按体表面积换算成相应剂量,观察股iv RIP1183后,对各组犬心血管系统和呼吸系统的影响.结果 与对照组相比,RIP1183对小鼠自主活动、协调运动能力和阈下巴比妥钠协同作用无影响,对比格犬心电图、心率、心律及呼吸频率和幅度、血压无明显影响.结论 RIP1183对中枢神经系统、呼吸系统和心血管系统均无明显影响.研究结果可为RIP1183临床试验研究提供参考.

Objective To investigate the effect of albendazole (ABZ) on tumor growth in nude mice model of glioma. Methods The glioblastoma tissue obtained during the operation was digested into single cell suspension and cultured in serum-free glioblastoma stem cell (GSC) medium. The glioma cell lines U87, U251, and U172 were cultured in DMEM medium containing 10% fetal bovine serum. All the cultured cells were treated with ABZ with a final concentration of 25, 50, 100, 200 ng/ml, respectively. The proliferation rates of cultured cells were detected by MTT assay. The nude mice model of glioma was established using subcutaneous injection of 0.2 ml (5× 106 cells) GSC and U87 cells suspension into the right armpit. Thirty nude mice models were randomly divided into 6 groups, 3 groups of GSC and 3 groups of U87. Each transplantation model was divided into model group (intraabdominal injection of equal volume DMSO), low dose ABZ group (intraabdominal injection of ABZ, 50 mg/kg) and high dose ABZ group (intraabdominal injection of ABZ, 100 mg/kg). The long diameter and short diameter of the tumor were measured regularly to calculate the tumor volume, and the mRNA and protein expressions of vascular endothelial growth factor (VEGF) in tumor tissues of nude mice were detected by PCR and immunoblotting, respectively. Results ABZ had a significant inhibitory effect on the growth of GSC, U87, U251 and A172 cells in a concentration-dependent manner, and the inhibitory effect was better when the concentration was more than 50 ng/ml. After intraabdominal injection of ABZ, the growth of tumor volume in high dose and low dose ABZ group was significantly slower than that in control group (P<0.05). The expression levels of VEGF mRNA and protein in high dose and low dose ABZ groups were significantly lower than those in control group (P<0.05). Conclusion ABZ can inhibit the growth of glioma in nude mice, which may be related to the inhibition of tumor cell proliferation and angiogenesis of tumor.

The DNA tetrahedron (Td), as one of the novel DNA-based nanoscale biomaterials, has been extensively studied because of its excellent biocompatibility and increased possibilities for decorating precisely. Although the use of Td in laboratories is well established, knowledge surrounding the factors influencing its preparation and storage is lacking. In this research, we investigated the role of the magnesium ions, which greatly affect the structure and stability of DNA. We assembled 1, 2, 5, 10 and 20 μM Td in buffers containing different Mg2+ concentrations, demonstrating that 2 and 5 mM Mg2+ is optimal in these conditions, and that yields decrease dramatically once the DNA concentration reaches 20 μM or the Mg2+ concentration is lower than 0.5 mM. We also verified that the Td structure is retained better through freeze-thawing than lyophilization. Furthermore, a lower initial Mg2+ (≤2 mM) benefited the maintenance of Td structure in the process of lyophilization. Hence, our research sheds light on the influence of Mg2+ in the process of preparing and storing Td, and also provides some enlightenment on improving yields of other DNA nanostructures.

Discovery and development of new antibacterial drugs against multidrug resistant bacterial strains have become more and more urgent. Antisense oligonucleotides (ASOs) show immense potential to control the spread of resistant microbes due to its high specificity of action, little risk to human gene expression, and easy design and synthesis to target any possible gene. However, efficient delivery of ASOs to their action sites with enough concentration remains a major obstacle, which greatly hampers their clinical application. In this study, we reviewed current progress on delivery strategies of ASOs into bacteria, focused on various non-virus gene vectors, including cell penetrating peptides, lipid nanoparticles, bolaamphiphile-based nanoparticles, DNA nanostructures and Vitamin B12. The current review provided comprehensive understanding and novel perspective for the future application of ASOs in combating bacterial infections.

Given its high resistance, enhanced virulence, and high transmissibility, community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) pneumonia is highly associated with high morbidity and mortality. Anti-virulence therapy is a promising strategy that bypasses the evolutionary pressure on the bacterium to develop resistance. RNAIII-inhibiting peptide (RIP), as an accessory gene regulator ( agr )-specific inhibitor, significantly restricts the virulence of S. aureus and protects infected mice from death by blocking the agr quorum sensing system. The protective effects of RIP on the neutropenic mice completely disappeared in a neutrophil-deleted mouse infection model, but not in the macrophage-deleted mice. This result confirmed that the in vivo antibacterial activity of RIP is highly associated with neutrophil function. Phenol-soluble modulins (PSMs), as major leukocyte lysis toxins of CA-MRSA, are directly regulated by the agr system. In this experiment, PSMα1, 2, and 3 significantly induced neutrophil necroptosis by activating mixed lineage kinase-like protein (MLKL) phosphorylation and increasing lactate dehydrogenase release. The S. aureus supernatants harvested from the agr or psmα mutant strains both decreased the phosphorylation level of MLKL and cell lysis. PSMα1-mediated neutrophil lysis was significantly inhibited by necrosulfonamide, necrostatin-1, TNFα antibody, and WRW4. These results showed PSMα1 induced necroptosis depends on formylpeptide receptor 2 (FPR2)-mediated autocrine TNFα. Moreover, the neutrophil necroptosis induced by S. aureus was significantly suppressed and pneumonia was effectively prevented by the blockage of agrA and psmα expression levels. These findings indicate that PSMα-induced necroptosis is a major cause of lung pathology in S. aureus pneumonia and suggest that interfering with the agr quorum sensing signaling pathway is a potential therapeutic strategy.