Designer broad-spectrum polyimidazolium antibiotics

For a myriad of different reasons most antimicrobial peptides (AMPs) have failed to reach clinical application. Different AMPs have different shortcomings including but not limited to toxicity issues, potency, limited spectrum of activity, or reduced activity in situ. We synthesized several cationic peptide mimics, main-chain cationic polyimidazoliums (PIMs), and discovered that, although select PIMs show little acute mammalian cell toxicity, they are potent broad-spectrum antibiotics with activity against even pan-antibiotic-resistant gram-positive and gram-negative bacteria, and mycobacteria. We selected PIM1, a particularly potent PIM, for mechanistic studies. Our experiments indicate PIM1 binds bacterial cell membranes by hydrophobic and electrostatic interactions, enters cells, and ultimately kills bacteria. Unlike cationic AMPs, such as colistin (CST), PIM1 does not permeabilize cell membranes. We show that a membrane electric potential is required for PIM1 activity. In laboratory evolution experiments with the gram-positive Staphylococcus aureus we obtained PIM1-resistant isolates most of which had menaquinone mutations, and we found that a site-directed menaquinone mutation also conferred PIM1 resistance. In similar experiments with the gram-negative pathogen Pseudomonas aeruginosa, PIM1-resistant mutants did not emerge. Although PIM1 was efficacious as a topical agent, intraperitoneal administration of PIM1 in mice showed some toxicity. We synthesized a PIM1 derivative, PIM1D, which is less hydrophobic than PIM1. PIM1D did not show evidence of toxicity but retained antibacterial activity and showed efficacy in murine sepsis infections. Our evidence indicates the PIMs have potential as candidates for development of new drugs for treatment of pan-resistant bacterial infections.

AMPs and AMP mimics have attracted considerable attention as candidates for therapeutic development (1). The basic design elements include a region of charged residues, generally cationic residues, enabling interaction with bacterial cell surfaces, combined with a hydrophobic nature in AMPs (2). Unfortunately, AMPs and related polymers, in general, have one or more issues that limit their use as broad-spectrum antibiotics. Some are quite toxic to human cells, the potency of some is not adequate for human administration, others are sensitive to salt at levels present in human fluids, and some are too difficult and expensive to synthesize (3, 4). One broad-spectrum antimicrobial peptide, CST has seen increased recent use as a last resort antibiotic. CST is believed to kill bacteria by virtue of its ability to disrupt membrane integrity (5). This antibiotic requires intravenous administration and is nephrotoxic (6). The emergence of CST-resistant pathogens has also become a significant problem (7). We are unaware of any new broad-spectrum AMPs that have advanced to clinical trials.Imidazolium (IM) salts are antimicrobials (8), and there is an emerging literature on antimicrobial activity of side-chain and main-chain polyimidazolium (PIM) salts with chemical structures that are in some ways similar to those we describe. Although PIMs are potent antimicrobials, there are biocompatibility problems hindering their development, and some have somewhat limited activity spectra. As with other AMPs, there have been toxicity issues, potency issues, and delivery issues as many have large molecular masses, and there is little known about mammalian cell toxicity or mechanism of action (9–12).Here we show that members of a series of PIMs we designed and synthesized are potent broad-spectrum antibacterial compounds. We selected two for further analysis and showed they retain activity even against pan-antibiotic-resistant bacteria. Unlike CST and many other AMPs, which disrupt bacterial membranes, our model PIM is bactericidal without disrupting bacterial membranes. Our experiments provide insights about mechanism of action, the potential for the emergence of PIM resistance, and indicate PIMs are effective against a model gram-negative and a model gram-positive pathogen in murine infection models.     

Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes.Flagellated bacteria have tailored their motility to diverse habitats. For example, the enteric model organisms Salmonella enterica serovar Typhimurium and Escherichia coli colonize animal digestive tracts and can reside outside a host, assembling flagella over their cell body to swim. However, a diverse spectrum of flagellar swimming ability is seen across the bacterial kingdom. Caulobacter crescentus inhabits low-nutrient freshwater environments where it swims using a high-efficiency flagellar motor (1, 2), whereas Vibrio species produce high-speed, sodium-driven polar flagella to capitalize on the high sodium gradient of their marine habitat (3). On the other hand, the ε-proteobacteria and spirochetes, many of which thrive exclusively in association with a host, have evolved characteristically rapid and powerful swimming capabilities that enable them to bore through mucous layers coating epithelial cells or between tissues. Indeed, the ε-proteobacteria Campylobacter jejuni and Helicobacter pylori are capable of continued swimming in high-viscosity media that immobilize E. coli or Vibrio cells (4–6), and similar behavior is observed for spirochetes (7, 8).Despite differences in the organisms’ swimming ability, the flagellar motor is composed of a conserved core of ∼20 structural proteins (9). The mechanism of flagellar motility is conserved (10), with torque generated by rotor and stator components (9). Stator complexes, heterooligomers of four motility A (MotA) and two motility B (MotB) proteins, are thought to form a ring that surrounds the axial driveshaft. Transmembrane helices of MotA and MotB form an ion channel, and MotB features a large periplasmic domain that binds peptidoglycan (11, 12) and the flagellar structural component, the P-ring (13). The stator complex couples ion flux to exertion of force on the cytoplasmic rotor ring (the C-ring), which transmits torque to the axial driveshaft (the rod), universal joint (the hook), and helical propeller (the filament), culminating in propulsion of the bacterium. Biophysical (14) and freeze-fracture (15) studies together with modeling (16) have proposed that a tight ring of ∼11 stator complexes dynamically assembles around the rod above the outer lobe of the C-ring in closely related Salmonella and E. coli motors (which we collectively refer to as the “enteric motor”). However, despite these conclusions, and although the structures observed in subtomogram averages have been proposed to be the stator complexes (17–19), the locations and stoichiometries of the stator complexes remain to be confirmed.How can we explain the wide diversity in flagellar swimming abilities in the context of a conserved core flagellar motor? Biophysical studies suggest that the source of the difference lies, at least in part, in variations in the mechanical output of the motors themselves. Torques of motors from different bacteria have been shown to range over an order of magnitude, and torque correlates with swimming speed and the ability of bacteria to propel themselves through different viscosities, indicating that adaptations are likely to be at the level of the motor itself. [Torque also varies within a single species, up to a maximum value, as a function of the number of stator complexes incorporated into the motor (14)]. For example, C. crescentus motors have been measured to produce torques of 350 pN⋅nm (2). Estimates for the torque of the enteric motor ranges from ∼1,300 to ∼2,000 pN⋅nm (20, 21). The ε-proteobacterium H. pylori has been estimated to swim with torque of 3,600 pN⋅nm (22), and spirochetes are capable of swimming with 4,000 pN⋅nm of torque (21, 23). Sodium-driven motor torques in Vibrio spp. have been measured between ∼2,000 and 4,000 pN⋅nm (24), depending on the magnitude of the sodium gradient. It is noteworthy, however, that an estimated sodium motive force in Vibrio spp. that is lower than the standard E. coli proton motive force nevertheless drives the Vibrio motor with higher torque than the E. coli motor (24, 25), further suggesting that torque differences likely exist at the level of the motor. However, the molecular mechanism by which different motors might produce different torques has not been investigated.The simplest scenario for tuning motor torque would be evolved adaptation of motor architecture. In support of this scenario, we recently showed that many motors have evolved additional structures not found in the well-studied enteric motors (18), and we observed that the C-ring radius varies among species (17, 18). One of the most widespread novel structures is a periplasmic basal disk directly beneath the outer membrane, often co-occurring with varied uncharacterized additional structures, which we collectively term “disk complexes.” Consistently, disk complexes have been seen only in motors that produce torque higher than that in E. coli or Salmonella. For example, the sodium-driven ∼2,000+ pN⋅nm torque motors of Vibrio species assemble a disk complex featuring a basal disk beneath the outer membrane (18) in addition to smaller H- and T-rings composed of FlgOT (flagella O, T) and MotXY (motility X, Y), respectively (26, 27). It has been shown that the T-ring interacts with stator complexes in Vibrio spp. (28), although the exact location and number of stator complexes in Vibrio spp. remains unclear. ε-Proteobacteria such as Helicobacter species, C. jejuni, and Wolinella succinogenes also assemble disk complexes composed of large basal disks beneath the outer membrane together with additional smaller disks (18, 29). Although these and other cases of additional disks have been reported (18, 30), their relation to flagellar function remains enigmatic, and it is unclear if these widespread disk complexes are homologous or analogous.In this study, we hypothesized that bacteria have tuned their swimming abilities by evolving structural adaptations to their flagellar motors that would result in altered torque generation. Using electron cryo-tomography and subtomogram averaging, we found that Vibrio polar γ-proteobacterial and Campylobacter ε-proteobacterial flagellar motors incorporate 13 and 17 stator complexes, respectively, compared with the ∼11 in enteric bacteria. In both cases, these stator complexes are scaffolded into wider stator rings relative to the enteric motor by components of their respective disk complexes. The wider C. jejuni stator ring is further reflected in a considerably wider rotor C-ring. Further analysis of the components of the Vibrio and C. jejuni disk complexes reveals that they share a core protein, FlgP, but each has acquired diverse additional components to form divergent disk-complex architectures. We conclude by showing that our structural data of wider stator rings featuring additional stator complexes can quantitatively account for the differences in torque between different flagellar motors.     

Role of polymer–drug conjugates in organ-specific delivery systems

Polymers have been utilized to deliver the drug to targeted site in controlled manner, achieving the high-therapeutic efficacy. Polymeric drug conjugates having variable ligands as attachments have been proved to be biodegradable, stimuli sensitive and targeted systems. Numerous polymeric drug conjugates having linkers degraded by acidity or intracellular enzymes or sensitive to over expressed groups of diseased organ/tissue have been synthesized during last decade to develop targeted delivery systems. Most of these organs have number of receptors attached with different cells such as Kupffer cells of liver have mannose-binding receptors while hepatocytes have asialoglycoprotein receptors on their surface which mainly bind with the galactose derivatives. Such ligands can be used for achieving high targeting and intracellular delivery of the drug. This review presents detailed aspects of receptors found in different cells of specific organ and ligands with binding efficiency to these specific receptors. This review highlights the need of further studies on organ-specific polymer–drug conjugates by providing detailed account of polymeric conjugates synthesized till date having organ-specific targeting.     

Tailor‐Made Antimicrobial/Antiviral Star Polymer via ATRP of Cyclodextrin and Guanidine‐Based Macromonomer

Novel star‐like polymers are prepared via atom transfer radical polymerization (ATRP) of polyhexamethylene guanidine hydrochloride (PHMG) macromonomer and acrylamide (AM) using β‐cyclodextrin (CD) with 8‐active and 5‐active sites as a macroinitiator. The resulting star‐like polymers are characterized by gel permeation chromatography (GPC) and ¹H NMR and are used for deactivating bacteria and viruses. It is found that star polymers with comparable amounts of PHMG possess excellent antimicrobial activity, which, however, strongly depends on the topological structure (i.e., the arm number and the monomer ratio) of the composing copolymers. The in vitro antibacterial activities of the synthesized polymers are investigated against Escherichia coli in terms of the minimum inhibitory concentration (MIC), whereas the antiviral activity of star copolymers is assessed via a plaque assay against non‐enveloped adenovirus (ADV). The results show that the highest antimicrobial activity is achieved by the star‐like copolymer with the monomer ratio of 20:3 (AM:PHGM, mol/mol), while the number of functional arms is fixed at 8. The incorporation of PHMG also renders the star copolymer highly antiviral, thus permitting it to be used as an effective antibacterial/antiviral agent for various applications.

     

Improved Electrical Performance of Poly(3‐hexylthiophene) Induced by Stable Doping with Polymer Dopants

A series of brominated polystyrenes (BPSs) are readily synthesized and blended as polymer dopants with the p‐type semiconducting polymer, poly(3‐hexylthiophene) (P3HT), to improve theirelectrical performance. The obtained P3HT/BPS blend films exhibit increased carrier concentration resulting from doping of P3HT by BPS, which leads to excellent electrical performance, including enhanced hole mobility and conductivity, and the conductivity or mobility increases with the bromination degree of BPS. Compared with conventional small molecular dopants, the polymer dopant, BPS, shows not only excellent doping stability, but also good solution processibility, which makes it a promising materials for fabrication of low cost, flexible, transparent, and high performance solution processable organic electronic devices.

     

眼眶骨折修复的人工材料的研究进展

眼眶骨折的治疗常需要植入修复材料,但修复材料的选择仍然是临床上一个争论性问题。目前有多种材料可以用于眶壁重建,但都有不同的并发症。与其他材料相比,人工材料更能缩短术中时间,并且用之不竭。其中可吸收人工材料还具有不可比拟的优势—即避免了不可吸收材料植入后终身都有发生并发症的风险。由于单个材料的优势有限,近年来相继出现的由两种或两种以上材料组成的复合材料开始频繁的应用于临床。本文旨在探讨用于眼眶骨折修复的各种人工材料的优势和局限性以及未来的发展方向。     

多聚物型可吸收冠状动脉支架的研究进展

新一代药物洗脱支架由可吸收多聚物为平台,具有与金属支架相当的的支撑力,以及良好的生物相容性.既可以暂时支撑管壁,抑制早期血栓形成及晚期新生内膜增生,还可作为良好的药物载体实现局部控制释放,达到有效防止支架置入后的血管急性闭塞和降低再狭窄率.     

Imaging of Ca2+ Transients in Endothelial Cells of Single Perfused Capillaries: Correlation of Peak [Ca2+]i with Sites of Macromolecule Leakage

Objective: To investigate the mechanisms responsible for variation in the macromolecular leakage (formation of localized leaky sites) in venular microvessels with increased permeability, we examined the hypothesis that cytoplasmic calcium concentration [Ca²⁺]_i, does not increase uniformly within microvessel endothelial cells. Methods: We loaded the endothelial cells forming the walls of venular microvessels in frog mesentery with fura-2, and imaged [Ca²⁺]_i using a cooled CCD camera. Results: Control [Ca²⁺]_i was close to 60 nM in all regions. Control permeability was uniformly low in all microvessels. Exposure to ionomycin (5 mM) increased [Ca²⁺]_i in a biphasic manner, but not uniformly. There was variation in both time to peak (bimodal distribution) and peak [Ca²⁺]_i (274 ± 13 nM; mean variation above or below the peak value was 110 nM). Raising extracellular calcium from 1.1 to 5 mM increased the mean variation of [Ca²⁺]_i about peak values. Extravascular leakage of fluorescently labeled albumin or low-density lipoproteins was most prominent at sites where increases in [Ca²⁺]_i were largest. Conclusions: These data indicate that variation in [Ca²⁺]_i within individual endothelial cells or groups of cells could account, at least in part, for the distribution of localized leakage sites for macromolecules in venular microvessels in the high-permeability state.     

Folding of a donor-acceptor polyrotaxane by using noncovalent bonding interactions

Mechanically interlocked compounds, such as bistable catenanes and bistable rotaxanes, have been used to bring about actuation in nanoelectromechanical systems (NEMS) and molecular electronic devices (MEDs). The elaboration of the structural features of such rotaxanes into macromolecular materials might allow the utilization of molecular motion to impact their bulk properties. We report here the synthesis and characterization of polymers that contain pi electron-donating 1,5-dioxynaphthalene (DNP) units encircled by cyclobis(paraquat-p-phenylene) (CBPQT(4+)), a pi electron-accepting tetracationic cyclophane, synthesized by using the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The polyrotaxanes adopt a well defined "folded" secondary structure by virtue of the judicious design of two DNP-containing monomers with different binding affinities for CBPQT(4+). This efficient approach to the preparation of polyrotaxanes, taken alongside the initial investigations of their chemical properties, sets the stage for the preparation of a previously undescribed class of macromolecular architectures.