Modulation of barnacle (Balanus amphitrite Darwin) cyprid settlement behavior by sulfobetaine and carboxybetaine methacrylate polymer coatings

Zwitterionic polymers such as poly(sulfobetaine methacrylate) (polySBMA) and poly(carboxybetaine methacrylate) (polyCBMA) have demonstrated impressive fouling-resistance against proteins and mammalian cells. In this paper, the effects of these surface chemistries on the settlement and behavior of an ubiquitous fouling organism, the cypris larva of the barnacle Balanus amphitrite (=Amphibalanus amphitrite), were studied in the laboratory. Conventional settlement assays and behavioral analysis of cyprids using Noldus Ethovision 3.1 demonstrated significant differences in settlement and behavior on different surfaces. Cyprids did not settle on the polySBMA or polyCBMA surfaces over the course of the assay, whereas settlement on glass occurred within expected limits. Individual components of cyprid behavior were shown to differ significantly between glass, polySBMA and polyCBMA. Cyprids also responded differently to the two zwitterionic surfaces. On polySBMA, cyprids were unwilling or unable to settle, whereas on polyCBMA cyprids did not attempt exploration and left the surface quickly. In neither case was toxicity observed. It is concluded that a zwitterionic approach to fouling-resistant surface development has considerable potential in marine applications.     

A novel biofilm channel for evaluating the adhesion of diatoms to non-biocidal coatings

Laboratory assessment of the adhesion of diatoms to non-toxic fouling-release coatings has tended to focus on single cells rather than the more complex state of a biofilm. A novel culture system based on open channel flow with adjustable bed shear stress values (0–2.4?Pa) has been used to produce biofilms of Navicula incerta. Biofilm development on glass and polydimethylsiloxane elastomer (PDMSe) showed a biphasic relationship with bed shear stress, which was characterised by regions of biofilm stability and instability reflecting cohesion between cells relative to the adhesion to the substratum. On glass, a critical shear stress of 1.3–1.4?Pa prevented biofilm development, whereas on PDMS, biofilms continued to grow at 2.4?Pa. Studies of diatom biofilms cultured on zwitterionic coatings using a bed shear stress of 0.54?Pa showed lower biomass production and adhesion strength on poly(sulfobetaine methacrylate) compared to poly(carboxybetaine methacrylate). The dynamic biofilm approach provides additional information to supplement short duration laboratory evaluations.     

Influence of sulfobetaines on the stability of the Citrobacterdiversus ULA-27 beta-lactamase.

The activity and stability of beta-lactamase from Citrobacter diversus ULA-27 have been investigated in the presence of different ionic and zwitterionic surfactants. All the sulfobetaine surfactants tested allow the enzyme to retain its full activity, but the best stabilizing effect is greatly dependent on their structure. Very little variations on the monomer headgroup can significantly reduce enzyme deactivation or speed up the loss of activity with respect to buffer alone. The whole hydrophobic/hydrophilic balance on the headgroup seems to have a determining role in preserving beta-lactamase activity and structure. The presence of zwitterionic surfactants stabilizes the protein conformation toward denaturation by urea and low-temperature inactivation. Similar experiments were performed in the presence of other two zwitterionic surfactants, an amine oxide, dimethylmyristylamine oxide (DMMAO) and a carboxybetaine, cetyldimethylammonium methanecarboxylate (CB1-16). The former stabilizes the enzyme even better than the sulfobetaines, the latter quickly deactivates it. Therefore, the factors responsible for beta-lactamase stabilization are dependent not only on the zwitterionic nature of the surfactant headgroup but also specific interactions between the surfactant and the protein may be important.     

Statistical optimization of solid state fermentation conditions for the enhanced production of thermoactive chitinases by mesophilic soil fungi using response surface methodology and their application in the reclamation of shrimp processing by-products

The mesophilic strains Aspergillus flavus CFR 10 and Fusarium oxysporum CFR 8 are potent producers of extracellular thermoactive chitinases (endo-chitinase and β-N-acetylhexosaminidase). Chitinases have a wide range of applications in many areas including reclamation of seafood processing chitinous by-products. In the present study, the interactive effects of four fermentation conditions on thermoactive chitinase production by solid state fermentation (SSF) using commercial wheat bran (CWB) was investigated employing response surface methodology (RSM). Further, these chitinases were applied for the preparation of N-acetyl chitooligosaccharides from shrimp chitin. Statistical optimization resulted in the production (unit/g initial dry substrate, U/g IDS) of 19.8 endo-chitinase and 649.0 β-N-acetylhexosaminidase activity by A. flavus CFR 10, and 17.5 endo-chitinase and 319.9 β-N-acetylhexosaminidase activity by F. oxysporum CFR 8. Activity of crude endo-chitinase and β-N-acetylhexosaminidase were found to be optimum at 62?±?1 °C in a wide pH range. Hydrolysis of colloidal chitin with crude chitinases produced the maximum N-acetyl chitooligosaccharides yield (mmol/l) of 10.4?±?0.28 at 6 h and 10.2?±?0.01 at 30 h post-reaction initiation, respectively, by the enzymes of A. flavus CFR 10 and F. oxysporum CFR 8. HPLC analysis revealed the presence of N-acetyl chitooligosaccharides with N-acetyl chitotriose as the main end product of the colloidal chitin hydrolysis. These results indicate the potential of mesophilic A. flavus CFR 10 and F. oxysporum CFR 8 in the production of thermoactive chitinases employing the economical SSF process using CWB as an ideal substrate, as well as the potential of these chitinases for the reclamation of abundant shrimp processing by-products and production of defined N-acetyl chitooligosaccharides.     

Dual-functional biomimetic materials: nonfouling poly(carboxybetaine) with active functional groups for protein immobilization

We introduce a dual-functional biocompatible material based on zwitterionic poly(carboxybetaine methacrylate) (polyCBMA), which not only highly resists protein adsorption/cell adhesion, but also has abundant functional groups convenient for the immobilization of biological ligands, such as proteins. The dual-functional properties are unique to carboxybetaine moieties and are not found in other nonfouling moieties such as ethylene glycol, phosphobetaine, and sulfobetaine. The unique properties are demonstrated in this work by grafting a polyCBMA polymer onto a surface or by preparing a polyCBMA-based hydrogel. PolyCBMA brushes with a thickness of 10-15 nm were grafted on a gold surface using the surface-initiated atom transfer radical polymerization method. Protein adsorption was analyzed using a surface plasmon resonance sensor. The surface grafted with polyCBMA very largely prevented the nonspecific adsorption of three test proteins, that is, fibrinogen, lysozyme, and human chorionic gonadotropin (hCG). The immobilization of anti-hCG on the surface resulted in the specific binding of hCG while maintaining a high resistance to nonspecific protein adsorption. Transparent polyCBMA-based hydrogel disks were decorated with immobilized fibronectin. Aortic endothelial cells did not bind to the polyCBMA controls, but appeared to adhere well and spread on the fibronectin-modified surface. With their dual functionality and biomimetic nature, polyCBMA-based materials are very promising for their applications in medical diagnostics, biomaterials/tissue engineering, and drug delivery.     

Consoramides A–C,New Zwitterionic Alkaloids from the Fungus Irpex consors

In our ongoing search for new secondary metabolites from fungi, a basidiomycete fungus Irpex consors was selected for mycochemical investigation, and three new zwitterionic alkaloids (1-3) and five known compounds (4-8) were isolated from the culture broth (16 l) of I. consors. The culture filtrate was fractionated by a series of column chromatography including Diaion HP-20, silica gel, and Sephadex LH-20, Sep-Pak C₁₈ cartridge, medium pressure liquid chromatography (MPLC), and high pressure liquid chromatography (HPLC) to yield eight compounds (1-8). The structures of the isolated compounds were elucidated by the interpretation of nuclear magnetic resonance (NMR) spectra and high-resolution mass spectrometry (HR-MS). Their antioxidant and antibacterial activities were examined. The zwitterionic structures of three new sesquiterpene alkaloids (1-3) were determined together with five known compounds identified as stereumamide E (4), stereumamide G (5), stereumamide H (6), stereumamide D (7), and sterostrein H (8). This is the first report of the zwitterionic alkaloids in the culture broth of I. consors. Three new zwitterionic alkaloids were named as consoramides A–C (1-3).     

The underestimated N-glycomes of lepidopteran species

BackgroundInsects are significant to the environment, agriculture, health and biotechnology. Many of these aspects display some relationship to glycosylation, e.g., in case of pathogen binding or production of humanised antibodies; for a long time, it has been considered that insect N-glycosylation potentials are rather similar and simple, but as more species are glycomically analysed in depth, it is becoming obvious that there is indeed a large structural diversity and interspecies variability.MethodsUsing an off-line LC-MALDI-TOF MS approach, we have analysed the N-glycomes of two lepidopteran species (the cabbage looper Trichoplusia ni and the gypsy moth Lymantria dispar) as well as of the commonly-used T. ni High Five cell line.ResultsWe detected not only sulphated, glucuronylated, core difucosylated and Lewis-like antennal fucosylated structures, but also the zwitterion phosphorylcholine on antennal GlcNAc residues, a modification otherwise familiar from nematodes; in L. dispar, N-glycans with glycolipid-like antennae containing α-linked N-acetylgalactosamine were also revealed.ConclusionThe lepidopteran glycomes analysed not only display core α1,3-fucosylation, which is foreign to mammals, but also up to 5% anionic and/or zwitterionic glycans previously not found in these species.SignificanceThe occurrence of anionic and zwitterionic glycans in the Lepidoptera data is not only of glycoanalytical and evolutionary interest, but is of biotechnological relevance as lepidopteran cell lines are potential factories for recombinant glycoprotein production.     

A nondetergent sulfobetaine improves protein unfolding reversibility in microcalorimetric studies

A nondetergent sulfobetaine (NDSB) was found to improve unfolding reversibility of several proteins by inhibiting heat-induced aggregation. As a consequence, ΔH_cal/ΔH_vH ratios were also improved to values close to 1 for a two-state unfolding. NDSB is effective in a wide range of pH values and especially at acidic pH generally used to calculate ΔC_p values by the Kirchhoff relation. The sulfobetaine also allows recording protein refolding by protecting the heat-induced unfolded state against aggregation.     

From Arylamine N-Acetyltransferase to Folate-Dependent Acetyl CoA Hydrolase: Impact of Folic Acid on the Activity of (HUMAN)NAT1 and Its Homologue (MOUSE)NAT2

Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH ₃ from acetyl Coenzyme A occurred concomitantly with the appearance of a CH ₃ peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme’s active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these enzymes and for improving chemotherapeutic approaches to pathological conditions including estrogen receptor-positive breast cancer.     

Benzothiazolyl Guanidines as Antibacterials

Some new N-p-chlorophenyl-N′-2-(substituted) benzothiazolyl-N″-alkyl guanidines have been synthesized. Several of these including some N-p-chlorophenyl-N′-2-(substituted)benzothiazolyl guanidines and N-p-chlorophenyl-N′-2-(substituted) benzothiazolyl-N″-methyl guanidines^* have been tested for their antibacterial activities against B. subtilis, S. aureus, S. typhi., E. coli and A. tumefaciens, and also for their antitubercular activity against M. tuberculosis (H37Rv).     

Synthesis of Benzothiazolylguanidines as Antituberculars and Antibacterials

The synthesis of twenty three new N-p-tolyl-N′-2-(substituted)benzothiazolyl-N″-alkyl-guanidines has been reported. Several of these including some N-p-tolyl-N′-2-(substitued)- benzothiazolylguanidines* have been evaluated for their antitubercular activity against M. tuberculosis (H 37 Rv) and also for their antibacterial activity. Among these N-p-tolyl-N′-2-(6-methoxy)benzothiazolylguanidine and N-p-tolyl-N′2-(6-methyUbenzothiazolyl-N″-methyl-guanidine are the most active as antituberculars but all the tested compounds are inactive as antibacterials.     

Woodchuck Hepatitis Virus Enhancer I and Enhancer II Are Both Involved in N-myc2 Activation in Woodchuck Liver Tumors

Direct activation of the N-myc2 oncogene by insertion of woodchuck hepatitis virus (WHV) DNA is a major oncogenic step in woodchuck hepatocarcinogenesis. We previously reported that WHV enhancer II (We2), which controls expression of the core/pregenome RNA, can also activate the N-myc2 promoter in hepatoma cell lines. To better define the integrated WHV regulatory sequences responsible for N-myc2 promoter activation in woodchuck liver tumors, we analyzed the structure and enhancer activity of a single viral integrant found at the win locus in tumor 2260T1 and mapping approximately 175 kb 3′ of N-myc2. This viral insert was made of 11 concatemerized WHV fragments, 5 of which overlapped with We2 sequences and 1 with WHV sequence homologous to that of hepatitis B virus enhancer I (We1). In transient transfection assays in hepatoma-derived cells, the We2 activator was found to be fully effective only when inserted in close proximity to the N-myc2 promoter whereas the We1 element by itself was apparently devoid of activity. In contrast, the 2260T1 viral insert exhibited a potent enhancer capacity that depended both on multimerized We2 and on We1 sequences. In a survey of different woodchuck hepatomas, both elements were commonly found within integrated viral sequences involved in long-range N-myc2 activation.     

Compound design guidelines for evading the efflux and permeation barriers of Escherichia coli with the oxazolidinone class of antibacterials: Test case for a general approach to improving whole cell Gram-negative activity

Previously we reported the results from an effort to improve Gram-negative antibacterial activity in the oxazolidinone class of antibiotics via a systematic medicinal chemistry campaign focused entirely on C-ring modifications. In that series we set about testing if the efflux and permeation barriers intrinsic to the outer membrane of Escherichia coli could be rationally overcome by designing analogs to reside in specific property limits associated with Gram-negative activity: i) low MW (<400), ii) high polarity (clogD_7.4 <1), and iii) zwitterionic character at pH 7.4. Indeed, we observed that only analogs residing within these limits were able to overcome these barriers. Herein we report the results from a parallel effort where we explored structural changes throughout all three rings in the scaffold for the same purpose. Compounds were tested against a diagnostic MIC panel of Escherichia coli and Staphylococcus aureus strains to determine the impact of combining structural modifications in overcoming the OM barriers and in bridging the potency gap between the species. The results demonstrated that distributing the charge-carrying moieties across two rings was also beneficial for avoidance of the outer membrane barriers. Importantly, analysis of the structure-permeation relationship (SPR) obtained from this and the prior study indicated that in addition to MW, polarity, and zwitterionic character, having ≤4 rotatable bonds is also associated with evasion of the OM barriers. These combined results provide the medicinal chemist with a framework and strategy for overcoming the OM barriers in GNB in antibacterial drug discovery efforts.     

Site-Specific N-Glycosylation of the S-Locus Receptor Kinase and Its Role in the Self-Incompatibility Response of the Brassicaceae

The S-locus receptor kinase SRK is a highly polymorphic transmembrane kinase of the stigma epidermis. Through allele-specific interaction with its pollen coat-localized ligand, the S-locus cysteine-rich protein SCR, SRK is responsible for recognition and inhibition of self pollen in the self-incompatibility response of the Brassicaceae. The SRK extracellular ligand binding domain contains several potential N-glycosylation sites that exhibit varying degrees of conservation among SRK variants. However, the glycosylation status and functional importance of these sites are currently unclear. We investigated this issue in transgenic Arabidopsis thaliana stigmas that express the Arabidopsis lyrata SRKb variant and exhibit an incompatible response toward SCRb-expressing pollen. Analysis of single- and multiple-glycosylation site mutations of SRKb demonstrated that, although five of six potential N-glycosylation sites in SRKb are glycosylated in stigmas, N-glycosylation is not important for SCRb-dependent activation of SRKb. Rather, N-glycosylation functions primarily to ensure the proper and efficient subcellular trafficking of SRK to the plasma membrane. The study provides insight into the function of a receptor that regulates a critical phase of the plant life cycle and represents a valuable addition to the limited information available on the contribution of N-glycosylation to the subcellular trafficking and function of plant receptor kinases.     

Selective antibacterial activity of the cationic peptide PaDBS1R6 against Gram-negative bacteria

Infections caused by Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa foremost among them, constitute a major worldwide health problem. Bioinformatics methodologies are being used to rationally design new antimicrobial peptides, a potential alternative for treating these infections. One of the algorithms used to develop antimicrobial peptides is the Joker, which was used to design the peptide PaDBS1R6. This study evaluates the antibacterial activities of PaDBS1R6 in vitro and in vivo, characterizes the peptide interaction to target membranes, and investigates the PaDBS1R6 structure in contact with mimetic vesicles. Moreover, we demonstrate that PaDBS1R6 exhibits selective antimicrobial activity against Gram-negative bacteria. In the presence of negatively charged and zwitterionic lipids the structural arrangement of PaDBS1R6 transits from random coil to α-helix, as characterized by circular dichroism. The tertiary structure of PaDBS1R6 was determined by NMR in zwitterionic dodecylphosphocholine (DPC) micelles. In conclusion, PaDBS1R6 is a candidate for the treatment of nosocomial infections caused by Gram-negative bacteria, as template for producing other antimicrobial agents.     

N-Myc Regulates Expression of Pluripotency Genes in Neuroblastoma Including lif,klf2, klf4, and lin28b

myc genes are best known for causing tumors when overexpressed, but recent studies suggest endogenous myc regulates pluripotency and self-renewal of stem cells. For example, N-myc is associated with a number of tumors including neuroblastoma, but also plays a central role in the function of normal neural stem and precursor cells (NSC). Both c- and N-myc also enhance the production of induced pluripotent stem cells (iPSC) and are linked to neural tumor stem cells. The mechanisms by which myc regulates normal and neoplastic stem-related functions remain largely open questions. Here from a global, unbiased search for N-Myc bound genes using ChIP-chip assays in neuroblastoma, we found lif as a putative N-Myc bound gene with a number of strong N-Myc binding peaks in the promoter region enriched for E-boxes. Amongst putative N-Myc target genes in expression microarray studies in neuroblastoma we also found lif and three additional important embryonic stem cell (ESC)-related factors that are linked to production of iPSC: klf2, klf4, and lin28b. To examine the regulation of these genes by N-Myc, we measured their expression using neuroblastoma cells that contain a Tet-regulatable N-myc transgene (TET21N) as well as NSC with a nestin-cre driven N-myc knockout. N-myc levels closely correlated with the expression of all of these genes in neuroblastoma and all but lif in NSC. Direct ChIP assays also indicate that N-Myc directly binds the lif promoter. N-Myc regulates trimethylation of lysine 4 of histone H3 in the promoter of lif and possibly in the promoters of several other stem-related genes. Together these findings indicate that N-Myc regulates overlapping stem-related gene expression programs in neuroblastoma and NSC, supporting a novel model by which amplification of the N-myc gene may drive formation of neuroblastoma. They also suggest mechanisms by which Myc proteins more generally contribute to maintenance of pluripotency and self-renewal of ESC as well as to iPSC formation.     

Zwitterionic Sulfobetaine Hydrogel Electrolyte Building Separated Positive/Negative Ion Migration Channels for Aqueous Zn‐MnO2 Batteries with Superior Rate Capabilities

Hydrogel electrolytes have attracted increasing attention due to their potential uses in the fabrication of flexible solid‐state batteries. However, the development of hydrogel electrolytes is still in the initial stage and the number of available strategies is limited. Ideally, the hydrogel electrolyte should exhibit suitable ionic conductivity rate, mechanical strength, and biocompatibility for safety. In this study, a zwitterionic sulfobetaine/cellulose hydrogel electrolyte is fabricated using raw materials from natural plants, which exhibits a good biocompatibility with mammalian cells. The intrinsic zwitterionic groups on sulfobetaine chains can provide separated ion migration channels for positive and negative ions, which largely facilitates electrolyte ion transport. A solid‐state Zn‐MnO₂ battery with a fabricated zwitterionic gel electrolyte exhibits a very high rate performance. It exhibits a specific capacity of 275 mA h ${\text{g}}_{{\text{MnO}}_2}^{?1}$ at 1 C. Even up to 30 C, a high capacity of 74 mA h ${\text{g}}_{{\text{MnO}}_2}^{?1}$ is maintained during the charging–discharging for up to 10 000 cycles. For wearable applications, the flexible solid‐state batteries can be used as reliable and portable sources to power different wearable electronics such as a commercial smart watch, electroluminescent panel, and color electroluminescence line, which shows their large potentials for use in next‐generation flexible and wearable battery technologies.     

Marine antifouling performance of polymer coatings incorporating zwitterions

Zwitterionic materials display antifouling promise, but their potential in marine anti-biofouling is still largely unexplored. This study evaluates the effectiveness of incorporating small quantities (0–20% on a molar basis) of zwitterions as sulfobetaine methacrylate (SBMA) or carboxybetaine methacrylate (CBMA) into lauryl methacrylate-based coatings whose relatively hydrophobic nature encourages adhesion of the diatom Navicula incerta, a common microfouling organism responsible for the formation of ‘slime’. This approach allows potential enhancements in antifouling afforded by zwitterion incorporation to be easily quantified. The results suggest that the incorporation of CBMA does provide a relatively minor enhancement in fouling-release performance, in contrast to SBMA which does not display any enhancement. Studies with coatings incorporating mixtures of varying ratios of the cationic monomer [2-(methacryloyloxy)ethyl]trimethylammonium chloride and the anionic monomer (3-sulfopropyl)methacrylate, which offer a potentially lower cost approach to the incorporation of anionic and cationic charge, suggest these monomers impart little significant effect on biofouling.     

Investigations into the membrane activity of arenicin antimicrobial peptide AA139

Arenicin-3 is an amphipathic β-hairpin antimicrobial peptide that is produced by the lugworm Arenicola marina. In this study, we have investigated the mechanism of action of arenicin-3 and an optimized synthetic analogue, AA139, by studying their effects on lipid bilayer model membranes and Escherichia coli bacterial cells. The results show that simple amino acid changes can lead to subtle variations in their interaction with membranes and therefore alter their pre-clinical potency, selectivity and toxicity. While the mechanism of action of arenicin-3 is primarily dependent on universal membrane permeabilization, our data suggest that the analogue AA139 relies on more specific binding and insertion properties to elicit its improved antibacterial activity and lower toxicity, as exemplified by greater selectivity between lipid composition when inserting into model membranes i.e. the N-terminus of AA139 seems to insert deeper into lipid bilayers than arenicin-3 does, with a clear distinction between zwitterionic and negatively charged lipid bilayer vesicles, and AA139 demonstrates a cytoplasmic permeabilization dose response profile that is consistent with its greater antibacterial potency against E. coli cells compared to arenicin-3.