Efficacy of a novel lantibiotic, CMB001, against MRSA.

OBJECTIVES: To evaluate the efficacy of a novel lantibiotic, CMB001, against MRSA biofilms in vitro and in an in vivo experimental model of bacterial infection. METHODS: Antibacterial activity of CMB001 was measured in vitro after its exposure to whole blood or to platelet-poor plasma. In vitro efficacy of CMB001 against a Staphylococcus aureus biofilm was studied using scanning electron microscopy. The maximum tolerable dose in mice was determined and a preliminary pharmacokinetic analysis for CMB001 was performed in mice. In vivo efficacy was evaluated in a neutropenic mouse thigh model of infection. RESULTS: CMB001 maintained its antibacterial activity in the presence of blood or plasma for up to 24 h at 37°C. CMB001 efficiently killed S. aureus within the biofilm by causing significant damage to the bacterial cell wall. The maximum tolerable dose in mice was established to be 10 mg/kg and could be increased to 30 mg/kg in mice pretreated with antihistamines. In neutropenic mice infected with MRSA, treatment with CMB001 reduced the bacterial burden with an efficacy equivalent to that of vancomycin. CONCLUSIONS: CMB001 offers potential as an alternative treatment option to combat MRSA. It will be of interest to evaluate the in vivo efficacy of CMB001 against infections caused by other pathogens, including Clostridioides difficile and Acinetobacter baumannii, and to expand its pharmacokinetic/pharmacodynamic parameters and safety profile.

Mechanism of action and epitopes of Clostridium difficile toxin B-neutralizing antibody bezlotoxumab revealed by X-ray crystallography.

The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a ß-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells.

Mobile sailing robot for automatic estimation of fish density and monitoring water quality.

INTRODUCTION: The paper presents the methodology and the algorithm developed to analyze sonar images focused on fish detection in small water bodies and measurement of their parameters: volume, depth and the GPS location. The final results are stored in a table and can be exported to any numerical environment for further analysis. MATERIAL AND METHOD: The measurement method for estimating the number of fish using the automatic robot is based on a sequential calculation of the number of occurrences of fish on the set trajectory. The data analysis from the sonar concerned automatic recognition of fish using the methods of image analysis and processing. RESULTS: Image analysis algorithm, a mobile robot together with its control in the 2.4 GHz band and full cryptographic communication with the data archiving station was developed as part of this study. For the three model fish ponds where verification of fish catches was carried out (548, 171 and 226 individuals), the measurement error for the described method was not exceeded 8%. SUMMARY: Created robot together with the developed software has features for remote work also in the variety of harsh weather and environmental conditions, is fully automated and can be remotely controlled using Internet. Designed system enables fish spatial location (GPS coordinates and the depth). The purpose of the robot is a non-invasive measurement of the number of fish in water reservoirs and a measurement of the quality of drinking water consumed by humans, especially in situations where local sources of pollution could have a significant impact on the quality of water collected for water treatment for people and when getting to these places is difficult. The systematically used robot equipped with the appropriate sensors, can be part of early warning system against the pollution of water used by humans (drinking water, natural swimming pools) which can be dangerous for their health.

Principal growth directions in development of the lateral root in Arabidopsis thaliana.

BACKGROUND AND AIMS: During lateral root development a new meristem is formed within the mother root body. The main objective of this work was to simulate lateral root formation in Arabidopsis thaliana and to study a potential role of the principal directions in this process. Lateral root growth is anisotropic, so that three principal directions of growth can be distinguished within the organ. This suggests a tensorial character of growth and allows for its description by means of the growth tensor method. METHODS: First features of the cell pattern of developing lateral roots were analysed in A. thaliana and then a tensorial model for growth and division of cells for this case was specified, assuming an unsteady character of the growth field of the organ. KEY RESULTS: Microscopic observations provide evidence that the principal directions of growth are manifested at various developmental stages by oblique cell walls observed in different regions of the primordium. Other significant features observed are atypically shaped large cells at the flanks of young apices, as well as distinct boundaries between the mother root and the primordium. Simulations were performed using a model for growth. In computer-generated sequences the above-mentioned features could be identified. An attempt was made to reconstruct the virtual lateral root that included a consideration of the formation of particular tissue types based on literature data. CONCLUSIONS: In the cell pattern of the developing lateral root the principal directions of growth can be recognized through occurrence of oblique cell divisions. In simulation the role of these directions in cell pattern formation was confirmed, only when cells divide with respect to the principal directions can realistic results be obtained.

Effect of mechanical stress on Zea root apex. I. Mechanical stress leads to the switch from closed to open meristem organization.

The effect of mechanical stress on the root apical meristem (RAM) organization of Zea mays was investigated. In the experiment performed, root apices were grown through a narrowing of either circular (variant I) or elliptical (variant II) shape. This caused a mechanical impedance distributed circumferentially or from the opposite sides in variant I and II, respectively. The maximal force exerted by the growing root in response to the impedance reached the value of 0.15 N for variant I and 0.08 N for variant II. Significant morphological and anatomical changes were observed. The changes in morphology depended on the variant and concerned diminishing and/or deformation of the cross-section of the root apex, and buckling and swelling of the root. Anatomical changes, similar in both variants, concerned transformation of the meristem from closed to open, an increase in the number of the cell layers at the pole of the root proper, and atypical oblique divisions of the root cap cells. After leaving the narrowing, a return to both typical cellular organization and morphology of the apex was observed. The results are discussed in terms of three aspects: the morphological response, the RAM reorganization, and mechanical factors. Assuming that the orientation of division walls is affected by directional cues of a tensor nature, the changes mentioned may indicate that a pattern of such cues is modified when the root apex passes through the narrowing, but its primary mode is finally restored.

Xanthan gum as a carrier for bacterial cell entrapment: Developing a novel immobilised biocatalyst.

Xanthan gum (XAN) is a widely used polysaccharide in various industries. Because of its unique properties, in this study, an attempt was made to adopt the procedure of xanthan gum cross-linking for the entrapment of bacterial cells that are able to biodegrade naproxen. The developed procedure proved to be completely neutral for Bacillus thuringiensis B1(2015b) cells, which demonstrated a survival rate of 99%. A negative impact of entrapment was noted for strain Planococcus sp. S5, which showed a survival rate in the 93-51% range. To achieve good mechanical properties of the composites, they were additionally hardened using polydopamine (PDA). XAN/PDA composites revealed a high stability in a wide range of pH, and their sorption capacity included both cationic and anionic molecules. Analysis of the survival rate during storage at 4 °C in 0.9% NaCl showed that, after 35 days, 98-99% of B1(2015b) and 47% of S5 cells entrapped in XAN/PDA remained alive. This study also presents the results of naproxen biodegradation conducted using XAN/PDA/B1(2015b) in a trickling filter with autochthonous microflora. Hence, owing to the significant acceleration of drug biodegradation (1 mg/L in 14 days) and the chemical oxygen demand removal, the entrapped B1(2015b) cells in XAN/PDA composites showed a promising potential in bioremediation studies and industrial applications.

Isolation, Characterization and Structure Elucidation of a Novel Lantibiotic From Paenibacillus sp.

We have isolated and characterized a novel antibacterial peptide, CMB001, following an extensive screening effort of bacterial species isolated from diverse environmental sources. The bacterium that produces CMB001 is characterized as a Gram (+) bacillus sharing approximately 98.9% 16S rRNA sequence homology with its closest match, Paenibacillus kyungheensis. The molecule has been purified to homogeneity from its cell-free supernatant by a three-step preparative chromatography process. Based on its primary structure, CMB001 shares 81% identity with subtilin and 62% with nisin. CMB001 is active mainly against Gram-positive bacteria and Mycobacteriaceae but it is also active against certain Gram-negative bacteria, including multi-drug resistant Acinetobacter baumannii. It retains full antibacterial activity at neutral pH and displays a low propensity to select for resistance among targeted bacteria. Based on NMR and mass spectrometry, CMB001 forms a unique 3D-structure comprising of a compact backbone with one α-helix and two pseudo-α-helical regions. Screening the structure against the Protein Data Bank (PDB) revealed a partial match with nisin-lipid II (1WCO), but none of the lantibiotics with known structures showed significant structural similarity. Due to its unique structure, resistance profile, relatively broad spectrum and stability under physiological conditions, CMB001 is a promising drug candidate for evaluation in animal models of bacterial infection.

Epitopes and Mechanism of Action of the Clostridium difficile Toxin A-Neutralizing Antibody Actoxumab.

The exotoxins toxin A (TcdA) and toxin B (TcdB) are produced by the bacterial pathogen Clostridium difficile and are responsible for the pathology associated with C. difficile infection (CDI). The antitoxin antibodies actoxumab and bezlotoxumab bind to and neutralize TcdA and TcdB, respectively. Bezlotoxumab was recently approved by the FDA for reducing the recurrence of CDI. We have previously shown that a single molecule of bezlotoxumab binds to two distinct epitopes within the TcdB combined repetitive oligopeptide (CROP) domain, preventing toxin binding to host cells. In this study, we characterize the binding of actoxumab to TcdA and examine its mechanism of toxin neutralization. Using a combination of approaches including a number of biophysical techniques, we show that there are two distinct actoxumab binding sites within the CROP domain of TcdA centered on identical amino acid sequences at residues 2162-2189 and 2410-2437. Actoxumab binding caused the aggregation of TcdA especially at higher antibody:toxin concentration ratios. Actoxumab prevented the association of TcdA with target cells demonstrating that actoxumab neutralizes toxin activity by inhibiting the first step of the intoxication cascade. This mechanism of neutralization is similar to that observed with bezlotoxumab and TcdB. Comparisons of the putative TcdA epitope sequences across several C. difficile ribotypes and homologous repeat sequences within TcdA suggest a structural basis for observed differences in actoxumab binding and/or neutralization potency. These data provide a mechanistic basis for the protective effects of the antibody in vitro and in vivo, including in various preclinical models of CDI.

Design and Purification of Subunit Vaccines for Prevention of Clostridium difficile Infection.

Clostridium difficile is a gram-positive bacterium responsible for a large proportion of nosocomial infections in the developed world. C. difficile secretes toxins A and B (TcdA and TcdB) and both toxins act synergistically to induce a spectrum of pathological responses in infected individuals ranging from pseudomembranous colitis to C. difficile-associated diarrhea. Toxins A and B have been actively investigated as components of prophylactic vaccine as well as targets for therapeutic intervention with antibodies. Expression of such toxins by recombinant technology is often difficult and may require special handling and adherence to strict safety regulations during the manufacturing process due to the inherent toxicity of the proteins. Both toxins are large proteins (308 kDa and 270 kDa, respectively) and contain distinct domains mediating cell attachment, cellular translocation, and enzymatic (glucosidase) activity. Here we describe methods to produce fragments of Toxin B for their subsequent evaluation as components of experimental C. difficile vaccines. Methods presented include selection of fragments encompassing distinct functional regions of Toxin B, purification methods to yield high quality proteins, and analytical evaluation techniques. The approach presented focuses on Toxin B but could be applied to the other component, Toxin A, and/or to any difficult to express or toxic protein.

SEM-EDS and X-ray micro computed tomography studies of skeletal surface pattern and body structure in the freshwater sponge Spongilla lacustris collected from Goczalkowice reservoir habit (Southern Poland).

INTRODUCTION: Freshwater sponges are common animals of most aquatic ecosystems. They feed by filtering small particles from the water, and so are thought to be sensitive indicators of pollution. Sponges are strongly associated with the abiotic environment and are therefore used as bioindicators for monitoring of water quality in water habitats. Among the freshwater sponges, Spongilla lacustris is one of the classic models used to study evolution, gene regulation, development, physiology and structural biology in animal water systems. It is also important in diagnostic of aquatic environments. The aim of this study was to characterize and visualize three-dimensional architecture of sponge body and measure skeleton elements of S. lacustris from Goczalkowice reservoir for identification purposes. MATERIAL AND METHODS: The scanning electron microscopy with an energy dispersive X-ray microanalysis (SEM- -EDS) and X-ray micro computed tomography (micro-CT) were used to provide non-invasive visualization of the three-dimensional architecture of Spongilla lacustris body. RESULTS: We showed that sponge skeleton was not homogeneous in composition and comprised several forms of skeleton organization. Ectosomal skeleton occurred as spicular brushes at apices of primary fibres with cementing spongin material. Choanosomal skeletal architecture was alveolate with pauci- to multispicular primary fibres connected by paucispicular transverse fibres, made by megascleres embedded in a scanty spongin matrix both in the choanosome and at the sponge surface. In contrast, microscleres were irregularly scattered in choanosome and skeletal surface. Furthermore, SEM-EDS studies showed that the distribution of silica in megascleres and microscleres was observed along the spicules and sponge surface areas. CONCLUSIONS: In conclusion, we showed that the combination of SEM-EDS and micro-CT microscopy techniques allowed obtaining a complete picture of the sponge spatial architecture.

Development of a recombinant toxin fragment vaccine for Clostridium difficile infection.

Clostridium difficile infection (CDI) is the major cause of antibiotic-associated diarrhea and pseudomembranous colitis, a disease associated with significant morbidity and mortality. The disease is mostly of nosocomial origin, with elderly patients undergoing anti-microbial therapy being particularly at risk. C. difficile produces two large toxins: Toxin A (TcdA) and Toxin B (TcdB). The two toxins act synergistically to damage and impair the colonic epithelium, and are primarily responsible for the pathogenesis associated with CDI. The feasibility of toxin-based vaccination against C. difficile is being vigorously investigated. A vaccine based on formaldehyde-inactivated Toxin A and Toxin B (toxoids) was reported to be safe and immunogenic in healthy volunteers and is now undergoing evaluation in clinical efficacy trials. In order to eliminate cytotoxic effects, a chemical inactivation step must be included in the manufacturing process of this toxin-based vaccine. In addition, the large-scale production of highly toxic antigens could be a challenging and costly process. Vaccines based on non-toxic fragments of genetically engineered versions of the toxins alleviate most of these limitations. We have evaluated a vaccine assembled from two recombinant fragments of TcdB and explored their potential as components of a novel experimental vaccine against CDI. Golden Syrian hamsters vaccinated with recombinant fragments of TcdB combined with full length TcdA (Toxoid A) developed high titer IgG responses and potent neutralizing antibody titers. We also show here that the recombinant vaccine protected animals against lethal challenge with C. difficile spores, with efficacy equivalent to the toxoid vaccine. The development of a two-segment recombinant vaccine could provide several advantages over toxoid TcdA/TcdB such as improvements in manufacturability.

Monitoring structure and activity of nitrifying bacterial biofilm in an automatic biodetector of water toxicity.

Automatic biodetector of water toxicity is a biosensor based on monitoring of catalytic activity of the nitrifying bacteria. To create a standardized biosensing system, development of the biofilm must be characterized to determine the prerequisites for its biological (biocatalytic) stability. In this paper, growth of biofilm comprising ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the open cellular polyurethane material polyurethane sponge bioreactor has been investigated. Dynamics of the biofilm formation was estimated using AOB and NOB metabolic activity and the volume occupied by these two types of bacteria in the biofilm. Spectrophotometry liquid ion chromatography and image cytometry were used, respectively, for these measurements. A mathematical model of the dynamics of biofilm formation was established. These data indicate that open cellular polyurethane material is a good basis for the immobilization of nitrifying bacteria. Moreover, growth of the biofilm leads to its stable structural form, whose biocatalytic activity (12.29 for AOB and 6.84 micromol min(-1) for NOB) is constant in the long term.

Automatic biodetector of water toxicity (ABTOW) as a tool for examination of phenol and cyanide contaminated water.

We describe an automatic biodetector for continuous monitoring of water toxicity (ABTOW). Construction of the ABTOW is based on natural ability of the biofilm formation to immobilize consortia of nitrifying bacteria (the sensing element) on the open cellular polyurethane foam as the support. Change of rates of oxygen consumption is used as an indicator of biocatalytic activity (nitrification) of the bacteria in response to xenobiotics. Owing to this design the ABTOW features stability long-term use, is inexpensive and simple in operation. The dynamics of ABTOW response is studied in details for phenol and cyanide as model toxins. These data indicate that the sensitivity was 3.5 µM for phenol and 0.19 µM for cyanide, respectively. The magnitudes of toxic effect were proportional to concentration whereas kinetics of the response is an indicator for the mechanism of toxicity. Similar methodology is applied to quantify toxicity of a range of heavy metals, herbicides and oxidative chain inhibitors. One may conclude that the presented biodetector provides a good sensitivity for continuous on-line monitoring of toxicity in water.

Reversal of acid-induced and inflammatory pain by the selective ASIC3 inhibitor, APETx2.

BACKGROUND AND PURPOSE: Inflammatory pain is triggered by activation of pathways leading to the release of mediators such as bradykinin, prostaglandins, interleukins, ATP, growth factors and protons that sensitize peripheral nociceptors. The activation of acid-sensitive ion channels (ASICs) may have particular relevance in the development and maintenance of inflammatory pain. ASIC3 is of particular interest due to its restricted tissue distribution in the nociceptive primary afferent fibres and its high sensitivity to protons. EXPERIMENTAL APPROACH: To examine the contribution of ASIC3 to the development and maintenance of muscle pain and inflammatory pain, we studied the in vivo efficacy of a selective ASIC3 inhibitor, APETx2, in rats. KEY RESULTS: Administration of APETx2 into the gastrocnemius muscle prior to the administration of low pH saline prevented the development of mechanical hypersensitivity, whereas APETx2 administration following low-pH saline was ineffective in reversing hypersensitivity. The prevention of mechanical hypersensitivity produced by acid administration was observed whether APETx2 was applied via i.m. or i.t. routes. In the complete Freund's adjuvant (CFA) inflammatory pain model, local administration of APETx2 resulted in a potent and complete reversal of established mechanical hypersensitivity, whereas i.t. application of APETx2 was ineffective. CONCLUSIONS AND IMPLICATIONS: ASIC3 contributed to the development of mechanical hypersensitivity in the acid-induced muscle pain model, whereas ASIC3 contributed to the maintenance of mechanical hypersensitivity in the CFA inflammatory pain model. The contribution of ASIC3 to established hypersensitivity associated with inflammation suggests that this channel may be an effective analgesic target for inflammatory pain states.

Analogs of MK-499 are differentially affected by a mutation in the S6 domain of the hERG K+ channel.

Drug-induced long QT syndrome has been principally ascribed to block of the cardiac hERG K(+) channel. Methanesulfonanilides, such as MK-499, E-4031 and dofetilide, are potent hERG antagonists that likely bind along the S6 helix within the inner vestibule of the pore. To further investigate these interactions, we broadly explored the structure-activity relationships of closely related analogs of MK-499 using a high-throughput ion flux assay, and evaluated in greater detail using patch-clamp electrophysiology. We observed that substitutions at the 4-position on the benzopyran ring significantly affected the potency of these analogs with the rank order of unsubstituted approximately ketone>amine>hydroxyl, implicating an important interaction at this position. We also evaluated the potency of these analogs on an S6 mutant of hERG (F656A) previously shown to significantly reduce the affinity for MK-499 and other known hERG antagonists (e.g. cisapride, terfenadine). In contrast to MK-499 (4-hydroxyl) and either the amine or unsubstituted analogs, the potency of the ketone analog was unaffected by this mutation suggesting that a compensatory interaction may be unveiled with the aromatic to apolar substitution, possibly through hydrogen bonding with Ser624 based on molecular modeling. More significantly, we found that this mutation rendered hERG susceptible to block in the closed-state by the smaller, unsubstituted analog, but not by MK-499 or larger analogs. Together these data suggest that interaction with Phe656 is not an absolute requirement for the binding of all methanesulfonanilide compounds, and that this residue may play a broader role in regulating access to the inner vestibule.

High-throughput analysis of drug binding interactions for the human cardiac channel, Kv1.5.

The voltage-gated potassium channel Kv1.5 is one of the key regulators of membrane potential repolarization in human atrial myocytes and is considered a potential drug target to treat atrial fibrillation. In this study we sought to determine molecular mechanism of action of DPO-1, a diphenylphosphine oxide derivative recently shown to terminate experimental atrial arrhythmia without affecting ventricular refractory period. In addition, we provided similar analysis for additional two small molecule blockers, representing different structural classes: cyclohexanones (PAC) and nor-triterpenoids (correolide). To rapidly identify the residues within the Kv1.5 channel critical for blocking activity of these molecules, two functional high-throughput ion channel assays were employed together with site-directed mutagenesis. Our study revealed that the residues critical for blocking activity of for DPO-1 include T480, localized at the outer mouth of the pore, and two residues along S6 helix: V505 and I508. The overlapping site was identified for PAC and included residues T480 and V505. In contrast to DPO-1, the I508A mutation resulted in only a modest reduction in the block of Kv1.5 by PAC (9-fold). Correolide, the largest molecule examined, made widespread interactions along the entire length of the pore (from T480 to V516). In summary, we have identified multiple residues involved in forming high affinity binding site for Kv1.5 blockers. Similar approaches of high-throughput ion channel technologies, combined with site-directed mutagenesis, may allow for parallel, rapid and accurate analysis of ion channel interactions with multiple compounds and could facilitate the design of more potent and selective ion channel blockers.

Synthesis of the first sulfur-35-labeled hERG radioligand.

The synthesis of the first high specific activity S-35-labeled hERG radioligand, [(35)S]MK-0499, for use in HTS assays of drug candidates for hERG interaction is described. The radioligand is prepared by [(35)S]sulfonylation of a high diastereomeric excess (de) aniline precursor prepared from unlabeled MK-0499.

Functional and pharmacological properties of canine ERG potassium channels.

We established HEK-293 cell lines that stably express functional canine ether-à-go-go-related gene (cERG) K(+) channels and examined their biophysical and pharmacological properties with whole cell patch clamp and (35)S-labeled MK-499 ([(35)S]MK-499) binding displacement. Functionally, cERG current had the hallmarks of cardiac delayed rectifier K(+) current (I(Kr)). Channel opening was time- and voltage dependent with threshold near -40 mV. The half-maximum activation voltage was -7.8 +/- 2.4 mV at 23 degrees C, shifting to -31.9 +/- 1.2 mV at 36 degrees C. Channels activated with a time constant of 13 +/- 1 ms at +20 mV, showed prominent inward rectification at depolarized potentials, were highly K(+) selective (Na(+)-to-K(+) permeability ratio = 0.007), and were potently inhibited by I(Kr) blockers. Astemizole, terfenadine, cisapride, and MK-499 inhibited cERG and human ERG (hERG) currents with IC(50) values of 1.3, 13, 19, and 15 nM and 1.2, 9, 14, and 21 nM, respectively, and competitively displaced [(35)S]MK-499 binding from cERG and hERG with IC(50) values of 0.4, 12, 35, and 0.6 nM and 0.8, 5, 47, and 0.7 nM, respectively. cERG channels had biophysical properties appropriate for canine action potential repolarization and were pharmacologically sensitive to agents known to prolong QT. A novel MK-499 binding assay provides a new tool to detect agents affecting ERG channels.

Discovery of a nonpeptidic small molecule antagonist of the human platelet thrombin receptor (PAR-1).

The synthesis and biological evaluation of a series of nonpeptidic small molecule antagonists of the human platelet thrombin receptor (PAR-1) are described. Optimization of the 5-amino-3-arylisoxazole lead resulted in an approximate 100-fold increase in potency. The most potent of these compounds (54) inhibits platelet activation with IC(50)s of 90 nM against the thrombin receptor agonist peptide (TRAP) and 510 nM against thrombin as the agonist. Further, antagonist 54 fully blocks platelet aggregation stimulated by 1 nM thrombin for 10 min.