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Antibiotic resistance is a growing concern in the medical field. Drug-susceptible infections are often treated with β-lactam antibiotics, which bind to enzymes known as penicillin-binding proteins (PBPs). When the PBPs are disabled, the integrity of the cell wall is compromised, leading to cell lysis. Resistance renders β-lactam antibiotics ineffective, and clinicians turn to be more effective, but often more toxic, antibiotics. An alternative approach is combining antibiotics with compounds that disable resistance mechanisms. Previously, we have shown that low-molecular-weight 600 Da branched polyethylenimine restores β-lactam susceptibility to Gram-positive and Gram-negative pathogens with antibiotic resistance. In this study, this approach is extended to the homodimers of 600 Da BPEI that have improved potentiation properties compared to monomers of 600 Da BPEI and 1200 Da BPEI. The homodimers are synthesized by linking two 600 Da BPEI molecules with methylenebisacrylamide (MBAA). The resulting product was characterized with FTIR spectroscopy

Carbapenem-Resistant Enterobacteriaceae (CRE) are emerging pathogens that cause variety of severe infections. CRE evade antibiotic treatments because these bacteria produce enzymes that degrade a wide range of antibiotics including carbapenems and β-lactams. The formation of biofilms aggravates CRE infections, especially in a wound environment. These difficulties lead to persistent infection and non-healing wounds. This creates the need for new compounds to overcome CRE antimicrobial resistance and disrupt biofilms. Recent studies in our lab show that 600 Da branched polyethyleneimine (BPEI) and its derivative PEG350-BPEI can overcome antimicrobial resistance and eradicate biofilms in methicillin-resistant S. aureus , methicillin-resistant S. epidermidis , P. aeruginosa , and E. coli . In this study, the ability of 600 Da BPEI and PEG350-BPEI to eradicate carbapenem-resistant Enterobacteriaceae bacteria and their biofilms is demonstrated. We show that both BPEI and PEG350-BPEI have anti-biofilm efficacy against CRE strains expressing Klebsiella pneumoniae carbapenemases (KPCs) and metallo-β-lactamases (MBLs), such as New Delhi MBL (NDM-1). Furthermore, our results illustrate that BPEI affects planktonic CRE bacteria by increasing bacterial length and width. These data demonstrate the multi-functional properties of 600 Da BPEI and PEG350-BPEI to reduce biofilm formation and mitigate virulence in carbapenem-resistant Enterobacteriaceae .

Biofilm formation is an adaptive resistance mechanism that pathogens employ to survive in the presence of antimicrobials.

The rise of life-threatening carbapenem-resistant Enterobacteriaceae (CRE) infections has become a critical medical threat. Some of the most dangerous CRE bacteria can produce enzymes that degrade a wide range of antibiotics, including carbapenems and β-lactams. Infections by CRE have a high mortality rate, and survivors can have severe morbidity from treatment with toxic last-resort antibiotics. CRE have mobile genetic elements that transfer resistance genes to other species. These bacteria also circulate throughout the healthcare system. The mobility and spread of CRE need to be curtailed, but these goals are impeded by having few agents that target a limited range of pathogenic CRE species. Against CRE possessing the metallo-β-lactamase NDM-1, Klebsiella pneumoniae ATCC BAA-2146 and Escherichia coli ATCC BAA-2452, the potentiation of meropenem and imipenem is possible with low-molecular weight branched polyethylenimine (600 Da BPEI) and its poly(ethylene glycol) (PEG)ylated derivative (PEG-BPEI) that has a low in vivo toxicity. The mechanism of action is elucidated with fluorescence assays of drug influx and isothermal calorimetry data showing the chelation of essential Zn2+ ions. These results suggested that 600 Da BPEI and PEG-BPEI may also improve the uptake of antibiotics and β-lactamase inhibitors. Indeed, the CRE E. coli strain is rendered susceptible to the combination of piperacillin and tazobactam. These results expand the possible utility of 600 Da BPEI potentiators, where previously we have demonstrated the ability to improve antibiotic efficacy against antibiotic resistant clinical isolates of Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis.

Advances in biotechnology to treat and cure human disease have markedly improved human health and the development of modern societies. However, substantial challenges remain to overcome innate biological factors that thwart the activity and efficacy of pharmaceutical therapeutics. Until recently, the importance of extracellular DNA (eDNA) in biofilms was overlooked. New data reveal its extensive role in biofilm formation, adhesion, and structural integrity. Different approaches to target eDNA as anti-biofilm therapies have been proposed, but eDNA and the corresponding biofilm barriers are still difficult to disrupt. Therefore, more creative approaches to eradicate biofilms are needed. The production of eDNA often originates with the genetic material of bacterial cells through cell lysis. However, genomic DNA and eDNA are not necessarily structurally or compositionally identical. Variations are noteworthy because they dictate important interactions within the biofilm. Interactions between eDNA and biofilm components may as well be exploited as alternative anti-biofilm strategies. In this review, we discuss recent developments in eDNA research, emphasizing potential ways to disrupt biofilms. This review also highlights proteins, exopolysaccharides, and other molecules interacting with eDNA that can serve as anti-biofilm therapeutic targets. Overall, the array of diverse interactions with eDNA is important in biofilm structure, architecture, and stability.

Bacterial biofilms, often impenetrable to antibiotic medications, are a leading cause of poor wound healing. The prognosis is worse for wounds with biofilms of antimicrobial-resistant (AMR) bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant S. epidermidis (MRSE), and multi-drug resistant Pseudomonas aeruginosa (MDR-PA). Resistance hinders initial treatment of standard-of-care antibiotics. The persistence of MRSA, MRSE, and/or MDR-PA often allows acute infections to become chronic wound infections. The water-soluble hydrophilic properties of low-molecular-weight (600 Da) branched polyethylenimine (600 Da BPEI) enable easy drug delivery to directly attack AMR and biofilms in the wound environment as a topical agent for wound treatment. To mitigate toxicity issues, we have modified 600 Da BPEI with polyethylene glycol (PEG) in a straightforward one-step reaction. The PEG–BPEI molecules disable β-lactam resistance in MRSA, MRSE, and MDR-PA while also having the ability to dissolve established biofilms. PEG-BPEI accomplishes these tasks independently, resulting in a multifunction potentiation agent. We envision wound treatment with antibiotics given topically, orally, or intravenously in which external application of PEG–BPEIs disables biofilms and resistance mechanisms. In the absence of a robust pipeline of new drugs, existing drugs and regimens must be re-evaluated as combination(s) with potentiators. The PEGylation of 600 Da BPEI provides new opportunities to meet this goal with a single compound whose multifunction properties are retained while lowering acute toxicity.

[Image: see text] Methicillin-resistant Staphylococcus aureus (MRSA) infections pose a serious threat worldwide. MRSA is the predominant species isolated from medical-device-related biofilm infections and chronic wounds. Its ability to form biofilms grants it resistance to almost all antibiotics on the market. Answering the call for alternative treatments, our lab has been investigating the efficacy of 600 Da branched polyethylenimine (BPEI) as a β-lactam potentiator against bacterial biofilms. Our previous study showed promise against methicillin-resistant Staphylococcus epidermidis biofilms. This study extends our previous findings to eradicate a more virulent pathogen: MRSA biofilms. Microtiter minimum biofilm eradication concentration models, crystal violet assays, and electron microscopy images show synergistic effects between BPEI and ampicillin as a two-step mechanism: step one is the removal of the extracellular polymeric substances (EPS) to expose individual bacteria targets, and step two involves electrostatic interaction of BPEI with anionic teichoic acid in the cell wall to potentiate the antibiotic.

Microbial biofilms are ubiquitous in nature, and they pose a serious threat to public health. Staphylococcus epidermidis is the most common clinical isolate from healthcare-and medical device-related biofilm infections. No antibiotic currently on the market can eradicate pathogenic biofilms, which contain complex defense mechanisms composed of slimelike extracellular polymeric substances. Understanding the need to develop alternative approaches, we examine 600 Da branched polyethylenimine (BPEI) against methicillin-resistant Staphylococcus epidermidis (MRSE) bio-films. Here, a microtiter biofilm model is used to test the synergistic effects between the two components of our combination treatment: BPEI and β-lactam antibiotics. Electron microscopy was used to confirm the growth of MRSE biofilms from the model. Minimum biofilm eradication concentration assays, crystal violet assays, and biofilm kill curves suggest that BPEI exhibits antibiofilm activity and can potentiate β-lactams to eradicate MRSE biofilms.

The rise of life-threatening carbapenem-resistant Enterobacteriaceae (CRE) infections has become a critical medical threat. Some of the most dangerous CRE bacteria can produce enzymes that degrade a wide range of antibiotics, including carbapenems and β-lactams. Infections by CRE have a high mortality rate, and survivors can have severe morbidity from treatment with toxic last-resort antibiotics. CRE have mobile genetic elements that transfer resistance genes to other species. These bacteria also circulate throughout the healthcare system. The mobility and spread of CRE need to be curtailed, but these goals are impeded by having few agents that target a limited range of pathogenic CRE species. Against CRE possessing the metallo-β-lactamase NDM-1, Klebsiella pneumoniae ATCC BAA-2146 and Escherichia coli ATCC BAA-2452, the potentiation of meropenem and imipenem is possible with low-molecular weight branched polyethylenimine (600 Da BPEI) and its poly(ethylene glycol) (PEG)ylated derivative (PEG-BPEI) that has a low in vivo toxicity. The mechanism of action is elucidated with fluorescence assays of drug influx and isothermal calorimetry data showing the chelation of essential Zn(2+) ions. These results suggested that 600 Da BPEI and PEG-BPEI may also improve the uptake of antibiotics and β-lactamase inhibitors. Indeed, the CRE E. coli strain is rendered susceptible to the combination of piperacillin and tazobactam. These results expand the possible utility of 600 Da BPEI potentiators, where previously we have demonstrated the ability to improve antibiotic efficacy against antibiotic resistant clinical isolates of Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis.

Infections from antibiotic resistant Staphylococcus aureus and Pseudomonas aeruginosa are a serious threat because reduced antibiotic efficacy complicates treatment decisions and prolongs the disease state in many patients. To expand the arsenal of treatments against antimicrobial resistant (AMR) pathogens, 600-Da branched polyethylenimine (BPEI) can overcome antibiotic resistance mechanisms and potentiate β-lactam antibiotics against Gram-positive bacteria. BPEI binds cell wall teichoic acids and disables resistance factors from penicillin binding proteins PBP2a and PBP4. The present study describes a new mechanism of action for BPEI potentiation of antibiotics generally regarded as agents effective against Gram-positive pathogens but not Gram-negative bacteria. 600-Da BPEI is able to reduce the barriers to drug influx and facilitate the uptake of a non-β-lactam co-drug, erythromycin, that targets the intracellular machinery. Also, BPEI can suppress production of the cytokine interleukin IL-8 by human epithelial keratinocytes. This enables BPEI to function as a broad-spectrum antibiotic potentiator which expands the opportunities to improve drug design, antibiotic development, and therapeutic approaches against pathogenic bacteria, especially for wound care.

Clinicians prescribe hundreds of millions of β-lactam antibiotics to treat the majority of patients presenting with bacterial infections. Patient outcomes are positive unless resistant bacteria, such as Pseudomonas aeruginosa (P. aeruginosa), are present. P. aeruginosa has both intrinsic and acquired antibiotic resistance, making clinical management of infection a real challenge, particularly when these bacteria are sequestered in biofilms. These problems would be alleviated if, upon the initial presentation of bacterial infection symptoms, clinicians were able to administer an antibiotic that kills both susceptible and otherwise resistant bacteria and eradicates biofilms. As the most common class of antibiotics, β-lactams could be used in a new drug if the leading causes of β-lactam antibiotic resistance, permeation barriers from lipopolysaccharide, efflux pumps, and β-lactamase enzymes, were also defeated. Against P. aeruginosa and their biofilms, the potency of β-lactam antibiotics is restored with 600 Da branched polyethylenimine (600 Da BPEI). Checkerboard assays using microtiter plates demonstrate the potentiation of piperacillin, cefepime, Meropenem, and erythromycin antibiotics. Growth curves demonstrate that only a combination of 600 Da BPEI and piperacillin produces growth inhibition antibiotic resistant P. aeruginosa. Scanning electron microscopy (SEM) was used to confirm that the combination treatment leads to abnormal P. aeruginosa morphology. Data collected with isothermal titration calorimetry and fluorescence spectroscopy demonstrate a mechanism of action in which potentiation at low concentrations of 600 Da BPEI reduces diffusion barriers from lipopolysaccharides without disrupting the outer membrane itself. Coupled with the ability to overcome a reduction in antibiotic activity created by biofilm exopolymers, targeting anionic sites on lipopolysaccharides and biofilm exopolysaccharides with the same compound provides new opportunities to counter the rise of multidrug-resistant infections.

Staphylococcus epidermidis is one of the most prevalent prokaryotic species on human skin and mucosal membranes that constitute the commensal flora. S. epidermidis has become one of the most common causes of primary bacteremia. Infections are difficult to diagnose because the pathogen has natural niches on human skin and the ability to adhere to inanimate surfaces via biofilms. Alarmingly, S. epidermidis has acquired resistance to many antibiotics, which presents a danger to human health. Known as methicillin-resistant S. epidermidis (MRSE), most clinical isolates of MRSE in North America exhibit β-lactam resistance primarily due to the presence of mecA, a gene that bestows β-lactam antibiotic resistance in a manner similar to methicillin-resistant Staphylococcus aureus (MRSA). MecA encodes for expression of penicillin-binding protein 2a (PBP2a), which is absent in β-lactam susceptible strains of S. epidermidis. We can disable this resistance factor in MRSE with 600-Da branched polyethylenimine (BPEI). Cationic BPEI targets anionic wall teichoic acid (WTA), an essential cofactor for proper functioning of PBP2a. We found that BPEI synergizes the activity of β-lactam antibiotics against MRSE. Growth curves suggest that the combination of BPEI and oxacillin is bactericidal. Electron micrographs indicate abnormalities in the cellular septa and cell walls of treated samples. Therefore, first-line clinical treatments can be effective against MRSE when used in combination with BPEI.

Methicillin-resistant Staphylococcus aureus (MRSA) is a medical concern. Here, we show that branched polyethylenimine (BPEI), a nontoxic, cationic polymer, restores MRSA’s susceptibility to β-lactam antibiotics. Checkerboard assays with MRSA demonstrated synergy between BPEI and β-lactam antibiotics. A time-killing curve showed BPEI to be bactericidal in combination with oxacillin. BPEI did not potentiate efficacy with vancomycin, chloramphenicol, or linezolid. When exposed to BPEI, MRSA increased in size and had difficulty forming septa. BPEI electrostatically binds to wall teichoic acid (WTA), a cell wall anionic polymer of Gram-positive bacteria that is important for localization of certain cell wall proteins. Lack of potentiation in a WTA knockout mutant supports the WTA-based mechanism. These data suggest that BPEI may prevent proper localization of cell wall machinery by binding to WTA; leading to cell death when administered in combination with β-lactam antibiotics. Negligible in vitro toxicity sug...

Polymers formed from N-isopropylacrylamide (NIPAM) are highly water soluble and undergo a temperature-induced phase transition to an insoluble state. The phase behavior is determined by competing hydrophilic and hydrophobic forces. In this report, additional insight regarding the effect soluble metals have on the phase transition process is provided by showing that cation solvation aids with stabilization of hydrophobic forces. This reduces barriers to rehydration and decreases thermodynamic entropy and enthalpy, obtained with variable-temperature (1)H nuclear magnetic resonance spectroscopy of NIPAM hydrogels in D(2)O, NaCl, MgCl(2), and CaCl(2). For the series of cations studied, it is observed that the order of increasing effect to facilitate the phase transition is Ca(2+) < Mg(2+) < Na(+). NaCl and MgCl(2) exhibited similar effects on the thermodynamics of the collapsing process. However, significant differences in the phase transition thermodynamics are observed between MgCl(2) and CaCl(2) salt solutions. The influence on Stage (1) enthalpy and entropy values for CaCl(2) solutions is approximately half that of the MgCl(2) solutions. This difference is likely related to their charge density of Ca(2+), which is approximately half that of Mg(2+).

Beta-lactam antibiotics kill Staphylococcus aureus bacteria by inhibiting the function of cell-wall penicillin binding proteins (PBPs) 1 and 3. However, β-lactams are ineffective against PBP2a, used by methicillin-resistant Staphylococcus aureus (MRSA) to perform essential cell wall crosslinking functions. PBP2a requires teichoic acid to properly locate and orient the enzyme, and thus MRSA is susceptible to antibiotics that prevent teichoic acid synthesis in the bacterial cytoplasm. As an alternative, we have used branched poly(ethylenimine), BPEI, to target teichoic acid in the bacterial cell wall. The result is restoration of MRSA susceptibility to the β-lactam antibiotic ampicillin with a MIC of 1 μg/mL, superior to that of vancomycin (MIC = 3.7 μg/mL). A checkerboard assay shows synergy of BPEI and ampicillin. Nuclear magnetic resonance (NMR) data show that BPEI alters the teichoic acid chemical environment. Laser scanning confocal microscopy (LSCM) images show BPEI residing on the bacterial cell wall where teichoic acids and PBPs are located.

Polymers formed from N-isopropylacrylamide (NIPAM) are highly water soluble and undergo a temperature-induced phase transition to an insoluble state. The phase behavior is determined by competing hydrophilic and hydrophobic forces. In this report, additional insight regarding the effect soluble metals have on the phase transition process is provided by showing that cation solvation aids with stabilization of hydrophobic forces. This reduces barriers to rehydration and decreases thermodynamic entropy and enthalpy, obtained with variable-temperature 1H nuclear magnetic resonance spectroscopy of NIPAM hydrogels in D2O, NaCl, MgCl2, and CaCl2. For the series of cations studied, it is observed that the order of increasing effect to facilitate the phase transition is Ca2+ < Mg2+ < Na+. NaCl and MgCl2 exhibited similar effects on the thermodynamics of the collapsing process. However, significant differences in the phase transition thermodynamics are observed between MgCl2 and CaCl2 salt solutions. The influence on Stage 1 enthalpy and entropy values for CaCl2 solutions is approximately half that of the MgCl2 solutions. This difference is likely related to their charge density of Ca2+, which is approximately half that of Mg2+.

Antifreeze proteins in fish, plants, and insects provide protection to a few degrees below freezing. Microbes have been found to survive at even lower temperatures, and with a few exceptions, antifreeze proteins are missing. We show that lipoteichoic acid (LTA), a biopolymer in the cell wall of Gram-positive bacteria, can be added to B. subtilis cultures and increase freeze tolerance. At 1% w/v, LTA enables a 50% survival rate, similar to the results obtained with 1% w/v glycerol as measured with the resazurin cell viability assay. In the absence of added LTA or glycerol, a very small number of B. subtilis cells survive freezing. This suggests that an innate freeze tolerance mechanism exists. While cryoprotection can be provided by extracellular polymeric substances (EPS), our data demonstrate a role for LTA in cryoprotection. Currently, the exact mode of action for LTA cryoprotection is unknown. With a molecular weight of 3-5 kDa, it is unlikely to enter the cell cytoplasm. However, low temperature microscopy data show small ice crystals aligned along channels of liquid water. Our observations suggest that teichoic acids could protect liquid water within biofilms and planktonic bacteria, augmenting the role of brine while also raising the possibility for survival without brine present.

Metals bind to the bacterial cell wall, yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membrane as lipoteichoic acid or covalently bound to the cell wall as wall teichoic acid. The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, which contradicts previous reports that calcium binding is 100 % dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger than previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, which means that the binding affinity decreases as more ions become bound to the sample. For Ca(2+), Region I has a KA = (1.0 ± 0.2) × 10(6) M(-1) and Region II has a KA = (0.075 ± 0.058) × 10(6) M(-1). For Mg(2+), KA1 = (1.5 ± 0.1) × 10(6) and KA2 = (0.17 ± 0.10) × 10(6). A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η2, which are 0.70 ± 0.04 and 0.67 ± 0.03 µmol/mg for Ca(2+) and Mg(2+) respectively. These data contradict the current paradigm of only a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent. This suggests a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations.

We found that different water environments in poly(N-isopropyl acrylamide) (PNIPAAm) hydrogels are identified and characterized using 1H high resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR). Local water environments corresponding to a “free” highly mobile species, along with waters showing restricted dynamics are resolved in these swollen hydro-gels. For photo-initiated polymerized PNIPAAm gels, an additional entrapped water species is observed. Spin–spin R2 relaxation experiments support the argument of reduced mobility in the restricted and entrapped water species. Furthermore, by combining pulse field gradient techniques with HRMAS NMR it is possible to directly measure the self-diffusion rate for these different water environments. The behavior of the heterogeneous water environments through the lower critical solution temperature transition is described.

Dormant bacterial spores are able to survive long periods of time without nutrients, withstand harsh environmental conditions, and germinate into metabolically active bacteria when conditions are favorable. Numerous factors influence this hardiness, including the spore structure and the presence of compounds to protect DNA from damage. It is known that the water content of the spore core plays a role in resistance to degradation, but the exact state of water inside the core is a subject of discussion. Two main theories present themselves: either the water in the spore core is mostly immobile and the core and its components are in a glassy state, or the core is a gel with mobile water around components which themselves have limited mobility. Using deuterium solid-state NMR experiments, we examine the nature of the water in the spore core. Our data show the presence of unbound water, bound water, and deuterated biomolecules that also contain labile deuterons. Deuterium-hydrogen exchange experiments show that most of these deuterons are inaccessible by external water. We believe that these unreachable deuterons are in a chemical bonding state that prevents exchange. Variable-temperature NMR results suggest that the spore core is more rigid than would be expected for a gel-like state. However, our rigid core interpretation may only apply to dried spores whereas a gel core may exist in aqueous suspension. Nonetheless, the gel core, if present, is inaccessible to external water.

High-resolution magic-angle-spinning NMR spectroscopy is used to investigate the phase transition of poly(N-isopropylacrylamide-co-acrylic acid) hydrogels crosslinked with N,N’-methylene bisacrylamide, hereafter poly(NIPAAm-co-AAc). Van't Hoff ΔH and ΔS for polymer dehydration are derived from temperature-dependent NMR spectra, and the thermodynamic data strongly support a four-stage dehydration mechanism for pure poly(NIPAAm). Acrylic acid stabilizes the hydration sphere around the polymer chains. Reduced amounts of water released during the phase transition translates into smaller values for ΔH and ΔS. Enhanced rehydration kinetics for poly(NIPAAm-co-AAc) is attributed to water remaining in the samples at elevated temperatures, which may produce facile diffusion pathways and enable faster rehydration kinetics than poly(NIPAAm).

NMR spectra were collected for poly(N-isopropylacrylamide) (PNIPAAm) hydrogel using high-resolution magic angle spinning (HRMAS) after gel pieces were hydrated in the presence of D2O, NaF, NaCl, and NaI aqueous solutions. Changes in the peak height intensity of the spectra provide quantitative insight into the phase transition process. The thermodynamic values of the phase transition were calculated using a van't Hoff analysis of the NMR data. Unlike the trend observed for decreases in the (LCST), changes in the enthalpy and entropy did not clearly display a linear dependence with respect to salt concentration. Rather, it was observed that increases in salt concentration did not affect the enthalpy and entropy to the extent as the initial change observed between no salt and 100 mM solutions. Finally, the effect of salts on the hysteresis of the rehydrating process was observed. Hysteresis occurs due to the need for hydrophobic interactions to break down before water is able to infiltrate the polymer matrix. NaF stabilizes hydrophobic interactions while NaI destabilize hydrophobic interactions, causing them to break down at higher temperatures. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012

An effective means of studying biological metal chemistry is through the use of cadmium NMR to probe the interaction between biomolecules, such as proteins and peptides, with divalent metals, such as zinc, copper, magnesium, or calcium. Gram-positive bacteria, such as S. aureus and B. subtilis , have peptidoglycan cell walls that contain teichoic acids, a poly(phosphodiester) biopolymer used for, among other things, metal chelation. Previous solid-state NMR and XAFS studies have shown that the cadmium ion binds in a bidentate manner to the phosphoryl centers of the dried teichoic acid backbone at physiological pH. However, current studies indicate that, when hydrated and at the low concentrations typically found in nature, the cadmium ions and phosphoryl sites interact through an extended solvent-separated ion pairing. These data reveal two unequal P-Cd interactions at distances of 4.2 and 4.9 A set approximately 180 degrees from each other in a linear arrangement.

Teichoic acids are important to bacteria for surface adhesion, metal ion coordination, and other biological processes crucial to bacterial survival. In particular, the surface adhesion of teichoic acids plays a crucial role in the formation of Gram-positive biofilms. Biofilms have been implicated as the major cause of various chronic infections. Biofilm formation is essentially a four-step process beginning with the adhesion of bacteria to a surface, followed by the excretion of an extracellular polymeric substance (slime), development and maturation of the biofilm architecture, and finally biofilm spreading through bacterial release. Currently, there is very little molecular level information available for the initial adhesion of bacteria to solid surfaces. Solid-state NMR is ideally suited for the study of these samples, thus we use 31 P solid-state NMR experiments to study the initial adhesion of lipoteichoic acid (LTA) to various surfaces. 31 P CP-MAS spectra and T 1 ρ data demonstrate that the structure of LTA changes when adhered to cellulose, cell wall peptidoglycan (PGN), or TiO 2 . However, when LTA is simultaneously adhered to PGN and TiO 2 the observed structure is dependent on the amount of retained water. For LTA on TiO 2 , we suggest that the alanine and glucosamine groups interact with the surface. However, during simultaneous adhesion to TiO 2 and PGN, the glucosamine groups bind to the PGN while the alanine groups bind to the surface. This arrangement traps water between the PGN and TiO 2 surface.

NMR spectra were collected for cross-linked poly(N-isopropylacrylamide), poly(NIPAM), hydrogels in the presence of NaCl and CaCl2 aqueous solutions. Intensity variations in the 1H NMR signals of the polymer provide insight into the phase transition process. These data were used to observe a two-stage phase transition process. Thermodynamic quantities were obtained from a van't Hoff analysis of the temperature-dependent equilibrium constants, which were derived from the NMR data. The Delta H degrees and Delta S degrees values for the hydrogel in D2O are 3.4 kJ/mol and 11.2 J/mol.K for stage I, which is attributed to the formation of hydrophobic bonds between neighboring isopropyl groups. The formation of hydrogen bonds during stage II yielded Delta H degrees and Delta S degrees values of 14.8 kJ/mol and 48.4 J/mol.K in D2O. However, the corresponding Delta H degrees values in 150 mM NaCl and 150 mM CaCl2 are reduced to 1.5 and 1.8 kJ/mol for stage I of the dehydration process. This corresponds to the known effect of salts on hydrophobic bond energetics. The value of Delta S degrees also decreased to 4.9 and 5.9 J/mol.K in NaCl and CaCl2 solutions, respectively. However, the thermodynamic values during stage II were only slightly affected by the salts. The lower temperatures required to induce spontaneous precipitation implies that Delta G degrees of precipitation is reduced. With our measurement of equilibrium thermodynamics, we see that 150 mM NaCl and CaCl2 solutions have a greater effect on hydrophobic bond formation associated with the phase transition process. In this manner, these salts aid in solvent reorganization necessary to form the hydrophobic bond, and this suggests that the formation of hydrophobic bonds is a strong determining factor in the stability of poly(NIPAM) hydrogels in water.

Bacterial spores can survive for long periods without nutrients and in harsh environmental conditions. This survival is influenced by the structure of the spore, the presence of protective compounds, and water retention. These compounds, and the physical state of water in particular, allow some species of bacterial spores to survive sterilization schemes with hydrogen peroxide and UV light. The chemical nature of the spore core and its water has been a subject of some contention and the chemical environment of the water impacts resistance paradigms. Either the spore has a glassy core, where water is immobilized along with other core components, or the core is gel-like with mobile water diffusion. These properties affect the movement of peroxide and radical species, and hence resistance. Deuterium solid-state NMR experiments are useful for examining the nature of the water inside the spore. Previous work in our lab with spores of Bacillus subtilis indicate that, for spores, the core water is in a more immobilized state than expected for the gel-like core theory, suggesting a glassy core environment. Here, we report deuterium solid-state NMR observations of the water within UV- and peroxide-resistant spores from Bacillus pumilus SAFR-032. Variable-temperature NMR experiments indicate no change in the line shape after heating to 50 °C, but an overall decrease in signal after heating to 100 °C. These results show glass-like core dynamics within B. pumilus SAFR-032 that may be the potential source of its known UV-resistance properties. The observed NMR traits can be attributed to the presence of an exosporium containing additional labile deuterons that can aid in the deactivation of sterilizing agents.

Estrogens are potent antioxidants and neuroprotectants. They have become the focus of drug development efforts aimed at treating and preventing neuronal damage in stroke-related brain damage, Alzheimer's disease, and Parkinson's disease. 13C{31P} and 13C{19F} rotational-echo double-resonance NMR have been used to map the locations of 17β-estradiol (estradiol), 17β-estradiol 17-benzoate (estradiol benzoate), and 17β-2-(1-adamantyl)estradiol (adamantyl estradiol) in 19F-labeled DPPC phospholipid multilamellar vesicles. Placement of the estrogen molecules in lipid bilayers is correlated with the differences in their neuroprotective potency. Drug Dev. Res. 66:99–102, 2006. © 2006 Wiley-Liss, Inc.

The production of hyperpolarized Xe via spin exchange with optically pumped Rb atoms was studied at 47 000 G under high-pressure conditions. From variable pressure and temperature studies, the spin exchange efficiency was found to be pressure dependent and lower than comparable low-field measurements. Spin exchange due to short binary collisions is expected to be pressure independent, and the cross section was measured to be 〈σν〉SE=6.0 (±0.1)×10−17 cm3 s−1. The pressure dependent component was attributed to the formation of Rb–Xe van der Waals (vdW) complexes. The rate constant for spin exchange in Rb–Xe vdW complexes was measured to be κ=1400 (±100) s−1 in He buffer gas. This value is comparable, though somewhat smaller than vdW rates measured previously for other buffer gases. While it is well known that spin exchange occurs readily in vdW complexes when B0 10 000 G is not accounted for in current theories. Modulation of the hyperfine interaction during the vdW comple...

Peptidoglycan and teichoic acids are the major cell-wall components of Gram-positive bacteria that obtain and sequester metal ions required for biochemical processes. Delivery of metals to the cytoplasmic membrane is aided by anionic binding sites within the peptidoglycan and along the phosphodiester polymer of teichoic acid. The interaction with metals is a delicate balance between the need for attraction and ion diffusion to the membrane. Likewise, metal chelation from the extracellular fluid must initially have strong binding energetics that weaken within the cell wall to enable ion release. We employed atomic absorption and equilibrium dialysis to measure the metal binding capacity and metal binding affinity of wall teichoic acid and Mg2+. The data show that Mg2+ binds to WTA with a 1:2 Mg2+ to phosphate ratio with a binding capacity of 1.27 μmol/mg. The affinity of Mg2+ to WTA was also found to be 41 × 103 M−1 at low metal concentrations and 1.3 × 103 M−1 at higher Mg2+ concentrations due to weakening electrostatic effects. These values are lower than the values describing Mg2+ interactions with peptidoglycan. However, the binding capacity of WTA is 4 times larger than peptidoglycan. External WTA initially binds metals with positive cooperativity but metal binding switches to negative cooperativity whereas interior WTA which binds metals with only negative cooperativity. The relevance of this work is to describe changes in metal binding behavior depending on environment. When metals are sparse, chelation is strong to ensure survival yet the binding weakens when essential minerals are abundant.

We present an analysis of the cross-relaxation dynamics between optically polarized 129 Xe and surface 13 C and 1 H spins in methanol adsorbed on a SiO 2 surface. Analytical expressions for the 13 C polarization are derived, which show that the 13 C polarization is determined by cross-relaxation with both 129 Xe and 1 H spins. The 1 H spins act as a polarization sink; however, saturation of the protons results in a further increase in signal intensity. Replacing the 1 H spin reservoir with 2 H increases the 13 C signal by a factor of 12 (25 as compared to room-temperature data). Further improvements are anticipated using higher-powered lasers.

The equilibrium isotope effects (EIE) for the one-electron transfer between pyrene and seven regioselectively deuteriated pyrene isotopic isomers in dimethylformamide with 0.1 M tetrabutylammonium hexafluorophosphate were measured electrochemically. These data correlate linearly with the free energies (ΔG°) obtained in tetrahydrofuran using electron paramagnetic resonance (EPR) techniques. However, the slope of the resulting line is not unity, and it indicates that the EIE in the DMF system is only two-thirds of that in the THF system. PM3 calculated ΔG°'s, which would correspond to the gas phase electron transfers, also correlate linearly with both sets of experimental data, but the predicted magnitudes of the EIE's are smaller than those observed experimentally by either technique. The nonunity slopes probably reflect slight differences in ion solvation and/or ion association parameters between the anion radicals of the isotopic isomers. No general relationship between the EIE and the charge on the hydr...

Numerous chemical additives lower the freezing point of water, but life at sub-zero temperatures is sustained by a limited number of biological cryoprotectants. Antifreeze proteins in fish, plants, and insects provide protection to a few degrees below freezing. Microbes have been found to survive at even lower temperatures, and with a few exceptions, antifreeze proteins are missing. Survival has been attributed to external factors, such as the high salt concentration of brine veins and adhesion to particulates or ice crystal defects. We have discovered an endogenous cryoprotectant in the cell wall of bacteria, lipoteichoic acid biopolymers. Adding 1% LTA to bacteria cultures immediately prior to freezing provides 50% survival rate, similar to the results obtained with 1% glycerol. In the absence of an additive, bacterial survival is negligible as measured with the resazurin cell viability assay. The mode of action for LTA cryoprotection is unknown. With a molecular weight of 3-5 kDa, it is unlikely to enter the cell cytoplasm. Our observations suggest that teichoic acids could provide a shell of liquid water around biofilms and planktonic bacteria, removing the need for brine veins to prevent bacterial freezing.

The bacterial spore is a formidable container of life, protecting the vital contents from chemical attack, antimicrobial agents, heat damage, UV light degradation, and water dehydration. The exact role of the spore components remains in dispute. Nevertheless, water molecules are important in each of these processes. The physical state of water within the bacterial spore has been investigated since the early 1930's. The water is found two states, free or bound, in two different areas, core and non-core. It is established that free water is accessible to diffuse and exchange with deuterated water and that the diffusible water can access all areas of the spore. The presence of bound water has come under recent scrutiny and has been suggested the water within the core is mobile, rather than bound, based on the analysis of deuterium relaxation rates. Using an alternate method, deuterium quadrupole-echo spectroscopy, we are able to distinguish between mobile and immobile water molecules. In the absence of rapid motion, the deuterium spectrum of D2O is dominated by a broad line, whose line shape is used as a characteristic descriptor of molecular motion. The deuterium spectrum of bacterial spores reveals three distinct features: the broad peak of immobilized water, a narrow line of water in rapid motion, and a signal of intermediate width. This third signal is assigned this peak from partially deuterated proteins with the spore in which N-H groups have undergone exchange with water deuterons to form N-D species. As a result of these observations, the nature of water within the spore requires additional explanation to understand how the spore and its water preserve life.

Origin of life theories have produced numerous schools of thought, two of which are termed RNA World and Snowball Earth. Complex organic molecules can be produced by RNA catalysis. This chemistry would be hindered by ice formation that would sequester moecules in the frozen water matrix. We present experimental data showing that RNA retains antifreeze properties that would enable the exchange of reactants/products of the catalytic reactions.

The conformation of d-alanine (d-Ala) groups of bacterial teichoic acid is a central, yet untested, paradigm of microbiology. The d-Ala binds via the C-terminus, thereby allowing the amine to exist as a free cationic NH(3)(+) group with the ability to form a contact ion pair with the nearby anionic phosphate group. This conformation hinders metal chelation by the phosphate because the zwitterion pair is charge neutral. To the contrary, the repulsion of cationic antimicrobial peptides (CAMPs) is attributed to the presence of the d-Ala cation; thus the ion pair does not form in this model. Solid-state nuclear magnetic resonance (NMR) spectroscopy has been used to measure the distance between amine and phosphate groups within cell wall fragments of Bacillus subtilis. The bacteria were grown on media containing (15)N d-Ala and beta-chloroalanine racemase inhibitor. The rotational-echo double-resonance (REDOR) pulse sequence was used to measure the internuclear dipolar coupling, and the results demonstrate (1) the metal-free amine-to-phosphate distance is 4.4 A and (2) the amine-to-phosphate distance increases to 5.4 A in the presence of Mg(2+) ions. As a result, the zwitterion exists in a nitrogen-oxygen ion pair configuration providing teichoic acid with a positive charge to repel CAMPs. Additionally, the amine of d-Ala does not prevent magnesium chelation in contradiction to the prevailing view of teichoic acids in metal binding. Thus, the NMR-based description of teichoic acid structure resolves the contradictory models, advances the basic understanding of cell wall biochemistry, and provides possible insight into the creation of new antibiotic therapies.

Recent studies from our lab demonstrated that teichoic acid is surrounded by liquid water at -40 °C. The size and shape of the liquid water pockets has been visualized with fluorescence microscopy images of aqueous Rhodamine- B solutions. The long, thin channels surround ice crystals with a size of 5-20 microns. Subsequent studies show that B. subtilis Gram-positive bacteria are sequestered into large pockets without added teichoic acid. Here, the ice crystals are orders of manitude larger. When bacteria are mixed with teichoic acid solutions, the distribution of bacteria changes dramatically. The smaller ice crystals allow the bacteria to align in the thin channels of liquid water seen with teichoic acid only. The role of teichoic acid in the freeze tolerance was examined with live/dead fluorescence assays of bacteria mixed with teichoic acid. These quantitative assays were used to determine if teichoic acid acts in a synergetic fashion to enhance the survivability of E. coli, a gram-negative species which lacks teichoic acid. Additionally, we have obtained B. subtilis mutants lacking wall-associated teichoic acids to evaluate cryoprotection compared to the wild-type strain.

Teichoic acids are essential components of the Gram-positive bacterial cell wall. One of their many functions is metal binding, a vital process for bacterial growth. With the combination of phosphorus-31 solid-state NMR spectroscopy and theoretical calculations using density functional theory (DFT), we have determined that the binding mode between teichoic acids and magnesium involves bidentate coordination by the phosphate groups of teichoic acid. Measurement of chemical shift anisotropy tensors gave a reduced anisotropy (delta) of 49.25 ppm and an asymmetry (eta) of 0.7. DFT calculations with diglycerol phosphate and triglycerol diphosphate model compounds were completed with Mg(2+) in anhydrous as well as partially hydrated bidentate and fully hydrated monodentate, bidentate, and bridging binding modes. (31)P CSA tensors were calculated from the energy-minimized model compounds using the combined DFT and GIAO methods, resulting in dramatically different tensor values for each binding mode. The anhydrous bidentate chelation mode was found to be a good approximation of the experimental data, an observation that alters the current monodentate paradigm for metal chelation by teichoic acids.

The high polarization of optically pumped 129Xe was transferred to surface carbon and silicon species under magic angle spinning (MAS) conditions to afford enhanced solid-state NMR of surfaces. High-resolution 29Si MAS spectra of fumed silica and 13C spectra of chemisorbed methanol on silica were obtained using a steady flow of hyperpolarized xenon adsorbed onto the surface at 135 K. Cross polarization to 29Si from SPINOE enhanced hydroxyl protons and to 13C from enhanced methyl protons is observed with good efficiency. A direct SPINOE transfer from 129Xe to 13C without cross polarization is observed to give the highest enhancement under these conditions.

Numerous chemical additives lower the freezing point of water, but life at sub-zero temperatures is sustained by a limited number of biological cryoprotectants. Antifreeze proteins in fish, plants, and insects provide protection to a few degrees below freezing. Microbes have been found to survive at even lower temperatures, although, with a few exceptions, antifreeze proteins are missing. Survival has been attributed to external factors, such as high salt concentration (brine veins) and adhesion to particulates or ice crystal defects. Teichoic acid is a phosphodiester polymer ubiquitous in Gram positive bacteria, composing 50% of the mass of the bacterial cell wall and excreted into the extracellular space of biofilm communities. We have found that when bound to the peptidoglycan cell wall (wall teichoic acid) or as a free molecule (lipoteichoic acid), teichoic acid is surrounded by liquid water at temperatures significantly below freezing. Using solid-state NMR, we are unable to collect 31 P CPMAS spectra for frozen solutions of lipoteichoic acid at temperatures above -60 °C. For wall teichoic acid in D 2 O, signals are not seen above -30 °C. These results can be explained by the presence of liquid water, which permits rapid molecular motion to remove 1 H/ 31 P dipolar coupling. 2 H quadrupole echo NMR spectroscopy reveals that both liquid and solid water are present. We suggest that teichoic acids could provide a shell of liquid water around biofilms and planktonic bacteria, removing the need for brine veins to prevent bacterial freezing.

Solid polymer electrolytes (SPEs) contain amorphous and crystalline regions, each of which have unique contributions to the (13)C NMR spectrum. Understanding and assigning the (13)C NMR signals are vital to interpreting the NMR data collected for each phase. The (13)C CPMAS solid-state NMR spectrum of poly(ethylene oxide), a common polymer electrolyte host material, has superimposed broad and narrow components. Previously, the narrow component has been assigned to the amorphous region and the broad component to the crystalline PEO fraction. These assignments for pure PEO have been applied to various PEO:salt systems. Using lithium triflate salt dissolved in PEO, we revisit the spectral assignments and discover that the narrow component is due to crystalline PEO:LiTf component, which is reversed from the previous pure PEO assignment. This paradigm shift is based on data collected from a 100% crystalline PEO:LiTf with a 3:1 oxygen:lithium ratio sample, which exhibited only the narrow peak. For dilute electrolytes, such as 20:1 PEO:LiTf, the (13)C CPMAS spectra contain the narrow peak superimposed on a broad peak as seen with pure PEO. As dilute electrolytes are heterogeneous with crystalline and amorphous regions of both pure PEO and PEO:LiTf complex, peak assignments for pure PEO and PEO:LiTf are important. Thus, we reexamine the previous assignment for pure PEO using samples of pure powdered PEO, thermally treated pure powdered PEO, and a thin film PEO cast from an acetonitrile solution. With these different samples, we observed the growth of the narrow peak under conditions that favor crystallization. Therefore, for pure PEO, we have reassigned the narrow peak to the crystalline region and the broad peak to the amorphous region. In light of our observations, previous NMR studies of pure PEO and PEO SPEs should be reinvestigated. We also use rotational echo double resonance (REDOR) to study the 20:1 PEO:LiTf created from 2 and 100 kDa PEO. We find that the lithium environment is similar in the respective microcrystalline domains. However, the 100 kDa samples have a larger fraction of pure crystalline PEO.

The role of the lithium ion environment is of fundamental interest regarding transport and conductivity in lithium polymer electrolytes. X-ray crystallography has been used to characterize the lithium environment in completely crystalline poly(ethylene oxide) (PEO) electrolytes, but this approach cannot be used with dilute PEO electrolytes. Here, using solid-state NMR data collected with the rotational-echo double-resonance 13C[7Li] (REDOR) pulse sequence, we have been able to characterize the crystalline microdomains of a PEO-lithium triflate sample with an oxygen/lithium ratio of 20:1. Our data clearly demonstrates that the lithium crystalline microdomains are nearly identical to those of a completely crystalline 3:1 sample, for which the crystal structure is known.

The adsorption of lipoteichoic acid onto the surface of titanium dioxide is shown to be heterogeneous. 31P CPMAS solid-state NMR reveals two distinct phosphate species. The chemical shift anisotropy, asymmetry parameter, and rotating-frame spin-lattice relaxation suggest that 50% of the phosphates are bound to the surface. The remaining phosphates also exhibit restricted molecular motion, but do not have a direct surface bond.

The solid anion radical salts resulting from the reductions of naphthalene, pyrene, perylene, benzo[k]fluoranthene, and biphenyl with lithium, cesium, and potassium metals were either pyrolyzed at 200-380 degrees C or irradiated with a pulsed frequency doubled Nd:YAG laser (532 nm wavelength). For both the pyrolytic and laser photolytic studies the only volatile products were hydrogen and methane. The hydrogen originated from the anionic polymerization of the anion radical. Evidence is presented that suggests that the methane results from a carbene intermediate, which in turn reacts with the H(2). Consistent with the H(2) formation, MALDS mass spectral analysis revealed the presence of dimers, trimers, and higher polymers of the polyaromatic hydrocarbon in the remaining nonvolatile products. There were considerably fewer low molecular weight oliomers produced via the laser photolysis than via the thermolysis.