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ConspectusIn this Account, we showcase site-directed Cu2+ labeling in proteins and DNA, which has opened new avenues for the measurement of the structure and dynamics of biomolecules using electron paramagnetic resonance (EPR) spectroscopy. In proteins, the spin label is assembled in situ from natural amino acid residues and a metal complex and requires no post-expression synthetic modification or purification procedures. The labeling scheme exploits a double histidine (dHis) motif, which utilizes endogenous or site-specifically mutated histidine residues to coordinate a Cu2+ complex. Pulsed EPR measurements on such Cu2+-labeled proteins potentially yield distance distributions that are up to 5 times narrower than the common protein spin label-the approach, thus, overcomes the inherent limitation of the current technology, which relies on a spin label with a highly flexible side chain. This labeling scheme provides a straightforward method that elucidates biophysical information that is costly, complicated, or simply inaccessible by traditional EPR labels. Examples include the direct measurement of protein backbone dynamics at β-sheet sites, which are largely inaccessible through traditional spin labels, and rigid Cu2+-Cu2+ distance measurements that enable higher precision in the analysis of protein conformations, conformational changes, interactions with other biomolecules, and the relative orientations of two labeled protein subunits. Likewise, a Cu2+ label has been developed for use in DNA, which is small, is nucleotide independent, and is positioned within the DNA helix. The placement of the Cu2+ label directly reports on the biologically relevant backbone distance. Additionally, for both of these labeling techniques, we have developed models for interpretation of the EPR distance information, primarily utilizing molecular dynamics (MD) simulations. Initial results using force fields developed for both protein and DNA labels have agreed with experimental results, which has been a major bottleneck for traditional spin labels. Looking ahead, we anticipate new combinations of MD and EPR to further our understanding of protein and DNA conformational changes, as well as working synergistically to investigate protein-DNA interactions.

Pseudomonas putida MPE exemplifies a novel clade of manganese-dependent single-strand DNA endonuclease within the binuclear metallophosphoesterase superfamily. MPE is encoded within a widely conserved DNA repair operon. Via structure-guided mutagenesis, we identify His113 and His81 as essential for DNA nuclease activity, albeit inessential for hydrolysis of bis-p-nitrophenylphosphate. We propose that His113 contacts the scissile phosphodiester and serves as a general acid catalyst to expel the OH leaving group of the product strand. We find that MPE cleaves the 3′ and 5′ single-strands of tailed duplex DNAs and that MPE can sense and incise duplexes at sites of short mismatch bulges and opposite a nick. We show that MPE is an ambidextrous phosphodiesterase capable of hydrolyzing the ssDNA backbone in either orientation to generate a mixture of 3′-OH and 3′-PO4 cleavage products. The directionality of phosphodiester hydrolysis is dictated by the orientation of the water nucleophile vis-à-vis the OH leaving group, which must be near apical for the reaction to proceed. We propose that the MPE active site and metal-bound water nucleophile are invariant and the enzyme can bind the ssDNA productively in opposite orientations.

There has been alarming depletion of manganese (Mn) reserves owing to the ongoing extensive mining operations for catering the massive industrial demand of this element. Moreover, the mining operations have been leading to the generation of Mn-rich waste, thereby contaminating both terrestrial and aquatic bodies. The current scenario necessitates the development of alternative processes for bioremediation as well as economic recovery of Mn from mining wastes. The present investigation aims to report the bioleaching of Mn by Lysinibacillus sp. from mining waste residues in the context of mine waste remediation. Results confirmed that the native isolate had a high Mn biosolubilization potential with a solubilizing efficiency of 84% at the end of a 21-day study under optimized conditions of pulp density 2% (

Mycobacterial Pol1 is a bifunctional enzyme composed of an N-terminal DNA flap endonuclease/5′ exonuclease domain (FEN/EXO) and a C-terminal DNA polymerase domain (POL). Here we document additional functions of Pol1: FEN activity on the flap RNA strand of an RNA:DNA hybrid and reverse transcriptase activity on a DNA-primed RNA template. We report crystal structures of the POL domain, as apoenzyme and as ternary complex with 3′-dideoxy-terminated DNA primer-template and dNTP. The thumb, palm, and fingers subdomains of POL form an extensive interface with the primer-template and the triphosphate of the incoming dNTP. Progression from an open conformation of the apoenzyme to a nearly closed conformation of the ternary complex entails a disordered-to-ordered transition of several segments of the thumb and fingers modules and an inward motion of the fingers subdomain—especially the O helix—to engage the primer-template and dNTP triphosphate. Distinctive structural features of mycobacterial Pol1 POL include a manganese binding site in the vestigial 3′ exonuclease subdomain and a non-catalytic water-bridged magnesium complex at the protein-DNA interface. We report a crystal structure of the bifunctional FEN/EXO–POL apoenzyme that reveals the positions of two active site metals in the FEN/EXO domain.

Pulsed electron paramagnetic resonance (EPR) based distance measurements using the recently developed Cu2+-DPA label present a promising strategy for measuring DNA backbone distance constraints. Herein we develop force field parameters for Cu2+-DPA in order to understand the features of this label at an atomic level. We perform molecular dynamics (MD) simulations using the force field parameters of Cu2+-DPA on four different DNA duplexes. The distance between the Cu2+ centers, extracted from the 2 μs MD trajectories, agrees well with the experimental distance for all the duplexes. Further analyses of the trajectory provide insight into the orientation of the Cu2+-DPA inside the duplex that leads to such agreement with experiments. The MD results also illustrate the ability of the Cu2+-DPA to report on the DNA backbone distance constraints. Furthermore, measurement of fluctuations of individual residues showed that the flexibility of Cu2+-DPA in a DNA depends on the position of the label in the duplex, and a 2 μs MD simulation is not sufficient to fully capture the experimental distribution in some cases. Finally, the MD trajectories were utilized to understand the key aspects of the double electron electron resonance (DEER) results. The lack of orientational selectivity effects of the Cu2+-DPA at Q-band frequency is rationalized in terms of fluctuations in the Cu2+ coordination environment and rotameric fluctuations of the label linker. Overall, a combination of EPR and MD simulations based on the Cu2+-DPA labelling strategy can contribute towards understanding changes in DNA backbone conformations during protein–DNA interactions.

Electron spin resonance (ESR) or electron paramagnetic resonance (EPR) spectroscopy is an incisive technique for the characterization of paramagnetic centers in inorganic, bioinorganic, and organic molecules and materials. These measurements are enabled with the help of paramagnetic species, such as organic free radicals and ions, electronically excited states, and metal–ligand compounds, some of which with biological importance. In this experiment, analogues of histidine–copper coordination compounds are investigated to show students how ESR line shapes depend on the complexed copper ion electronic structure as well as how the spectral features elucidate the coordination environment of copper. These coordinated compounds play an important role in metal ion coordination in proteins and peptides. The experiment has two parts. First, three copper–imidazole complexes that mimic copper–histidine compounds in the human body are synthesized. Second, the ESR spectrum of each complex is obtained at 77 K from frozen chloroform/methanol and acetonitrile/methanol matrixes and used to elucidate the ligand arrangement of the Cu2+ ion and the effect on the unpaired electron density.

The formation of granuloma is one of the characteristic features of tuberculosis. Besides, rise in the concentration of serum amyloid A (SAA) protein is the indicator for chronic inflammation associated with tuberculosis. The predisposition of SAA driven secondary amyloidosis in tuberculosis is well documented. However, SAA derived amyloid onset and deposition start sites are not well chracterised in tuberculosis and other chronic inflammatory conditions. We hypothesized that granuloma could be a potential site for amyloid deposition because of the presence of SAA protein and proteases that cleave SAA into aggregation prone fragments. 150 tuberculosis patients were screened and biopsies were collected from the affected organs of these patients. 20 patients showed eosinophilic hyaline rich deposits within and surrounding granuloma respectively. They were further screened for the presence of amyloid deposits. The hyaline material, upon Congo red staining exhibited characteristic apple green birefringence under polarized light, confirming deposition of amyloid. Further upon immuno histochemical staining with anti-SAA antibody, the amyloid enriched areas showed positive immunoreactivity. In this pilot study, wexx have shown granuloma as a potential site for serum amyloid A derived amyloid deposition in tuberculosis patients. This study would set a stage to expxand the clinical and fundamental research for understanding the mechanism of amyloid formation in granuloma underlying tuberculosis and other chronic inflammatory conditions.

BackgroundBiofilm associated infections are the major contributor of mortality, morbidity and financial burden in patients with bacterial infection. Molecules with surfactant behaviour are known to show significant antibiofilm effect against these infections. Thus, newly discovered antibacterial Fmoc-phenylalanine (Fmoc-F) and other Fmoc-amino acids (Fmoc-AA) with surfactant properties, could have potential antibiofilm properties.ObjectivesTo evaluate and characterise the antibiofilm activity of Fmoc-F and some Fmoc-AA against various clinically relevant bacteria.MethodsBiofilm inhibition and eradication was evaluated by crystal violet staining procedure along with scanning electron microscopy (SEM). Attenuated Total Reflection - Fourier Transform Infrared Spectroscopy (ATR-FTIR), Biochemical assays and Congo red staining were employed to investigate mechanism of antibiofilm action.ResultsWe showed that Fmoc-F not only inhibits the biofilm formation in S. aureus and P. aeruginosa, but also eradicates the already formed biofilms over the surface. Further, Fmoc-F coated glass surface resists S. aureus and P. aeruginosa biofilm formation and attachment, when biofilm is grown over the surface. The mechanistic investigation suggests that Fmoc-F reduces the ECM components such as proteins carbohydrates and eDNA in the biofilm and affect its stability via direct interactions with ECM components and/ or indirectly through reducing bacterial cell population. Finally, we showed that Fmoc-F treatment in combination with other antibiotics such as vancomycin and ampicillin synergistically inhibit biofilm formation.ConclusionsOverall, the study demonstrates the potential application of Fmoc-F and other Fmoc-AA molecules individually as well as in combination as antibiofilm agents and antibiofilm coating material for treating biofilm associated infections.

The interactions between proteins and their specific DNA sequences are the basis of many cellular processes. Hence, developing methods to build an atomic level picture of these interactions helps improve our understanding of key cellular mechanisms. CueR is an Escherichia coli copper-sensing transcription regulator. The inhibition of copper-sensing transcription regulators can kill pathogens, without harming the host. Several spectroscopic studies and crystallographic data have suggested that changes in the conformation of both the DNA and the protein control transcription. However, due to the inadequate resolution of these methods, the exact number of active conformations of CueR has not been determined. Resolving the structure of CueR in its active state is highly important for the development of specific inhibitors. Herein, the potential of double-histidine (dHis)-based CuII spin labeling for the identification of various conformational states of CueR during transcription is shown.

This paper reports an optical “TURN OFF” aptasensor, which is comprised of a deoxyribonucleic acid aptamer attached to a quantum dot on the $5^{\prime }$ terminus and gold nanoparticle on the $3^{\prime }$ terminus. The photoluminescence intensity is observed to decrease upon progressive addition of the target protein tumor necrosis factor-alpha (TNF- $\alpha $ ) to the sensor. For PBS-based TNF- $\alpha $ samples, the beacon exhibited 19%–20% quenching at around 22 nM concentration. The photoluminescence intensity and the quenching efficiency showed a linear decrease and a linear increase, respectively, between 0 to 22.3 nM TNF- $\alpha $ . The detection limit of the sensor was found to be 97.2 pM. Specificity test results determined that the sensor has higher selectivity toward TNF- $\alpha $ than other control proteins such as C—reactive protein, albumin, and transferrin. The beacon successfully detected different concentrations of TNF- $\alpha $ in human serum-based samples exhibiting around 10% quenching efficiency at 12.5 nM of the protein.

S100A12 or Calgranulin C is a homodimeric antimicrobial protein of the S100 family of EF-hand calcium-modulated proteins. S100A12 is involved in many diseases such as inflammation, tumor invasion, cancer and neurological disorders such as Alzheimer’s disease. The binding of transition metal ions to the protein is important as the sequestering of the metal ion induces conformational changes in the protein, inhibiting the growth of various pathogenic microorganisms. In this work, we probe the Cu2+ binding properties of Calgranulin C. We demonstrate that the two Cu2+ binding sites in Calgranulin C show different coordination environments in solution. Continuous wave-electron spin resonance (CW-ESR) spectra of Cu2+-bound protein clearly show two distinct components at higher Cu2+:protein ratios, which is indicative of the two different binding environments for the Cu2+ ions. The g|| and A|| values are also different for the two components, indicating that the number of directly coordinated nitrogen in each site differs. Furthermore, we perform CW-ESR titrations to obtain the binding affinity of the Ca2+-loaded protein to Cu2+ ions. We observe a positive cooperativity in binding of the two Cu2+ ions. To further probe the Cu2+ coordination, we also perform electron spin echo envelope modulation (ESEEM) experiment. We perform ESEEM at two different fields where one Cu2+ binding site dominates the other. At both sites we see distinct signatures of Cu2+–histidine coordination. However, we clearly see that the ESEEM spectra corresponding to the two Cu2+ binding sites are significantly different. There is clear change in the intensity of the double quantum peak with respect to the nuclear quadrupole interaction peak at the two different fields. Furthermore, ESEEM along with hyperfine sublevel correlation show that only one of the two Cu2+ binding sites has backbone coordination, confirming our previous observation. Finally, we perform double electron–electron resonance spectroscopy to probe if the difference in binding environment is due to the Cu2+ binding to different sites in the protein. We obtain a distance distribution with a sharp peak at ~ 3 nm and a broad peak at ~ 4 nm. The shorter distance agrees with the Cu2+–Cu2+ distance expected for a dimer from the crystal structure. The longer distance is consistent with the Cu2+–Cu2+ distance when oligomerization occurs.

Spin labels attached to two residues of a protein chain have less conformational flexibility than those attached to a single residue and thus lead to a narrower spatial distribution of the unpaired electron. The case of Cu2+ labels based on the double-histidine (dHis) motif is of particular interest, as it combines the advantage of precise localization of the unpaired electron with a labelling scheme orthogonal to the more common cysteine-based labelling. Here, we introduce an approach for in silico spin labelling of a protein by dHis motifs and Cu2+ complexes of iminodiacetic acid or nitrilotriacetic acid. We discuss a computerized scan for native histidine pairs that might be prone to bind such Cu2+ complexes and spin-labelling site pair scans that can identify suitable double mutants for labelling. Predicted distance distributions between two Cu2+ labels are compared to experimental distance distributions. We also test the hypothesis that elastic network modelling of conformational transitions with Cu2+-dHis labels can provide more accurate structural models than with nitroxide labels.

Site-directed spin labeling using two strategically placed natural histidine residues allows for the rigid attachment of paramagnetic Cu2+. This double histidine (dHis) motif enables extremely precise, narrow distance distributions resolved by Cu2+-based pulsed ESR. Furthermore, the distance measurements are easily relatable to the protein backbone-structure. The Cu2+ ion has, till now, been introduced as a complex with the chelating agent iminodiacetic acid (IDA) to prevent unspecific binding. Recently, this method was found to have two limiting concerns that include poor selectivity towards α-helices and incomplete Cu2+-IDA complexation. Herein, we introduce an alternative method of dHis-Cu2+ loading using the nitrilotriacetic acid (NTA)-Cu2+ complex. We find that the Cu2+-NTA complex shows a four-fold increase in selectivity toward α-helical dHis sites. Furthermore, we show that 100% Cu2+-NTA complexation is achievable, enabling precise dHis loading and resulting in no free Cu2+ in solution. We analyze the optimum dHis loading conditions using both continuous wave and pulsed ESR. We implement these findings to show increased sensitivity of the Double Electron-Electron Resonance (DEER) experiment in two different protein systems. The DEER signal is increased within the immunoglobulin binding domain of protein G (called GB1). We measure distances between a dHis site on an α-helix and dHis site either on a mid-strand or a non-hydrogen bonded edge-strand β-sheet. Finally, the DEER signal is increased twofold within two α-helix dHis sites in the enzymatic dimer glutathione S-transferase exemplifying the enhanced α-helical selectivity of Cu2+-NTA.

Acoustic phonon modes in a rectangular nanoscale waveguide deposited on a substrate (Siedel-White type waveguide) are analyzed analytically in terms of the elastic continuum model and the modes are quantized under the dimensional confinement assumption. The rectangular isotropic overlay structure is assumed to be deposited on a substrate with larger area compared to the overlay, and perfectly rigid. The displacement field inside the rectangular overlay structure is described for structures with nanometer dimensions. The frequency dependence of the modes has been studied along with the modal profiles for different overlay dimensions. The phonon occupation number has been evaluated for a range of dimensions and temperatures and the frequency-quality factor (f.Q) product has been theoretically discussed and evaluated using the parameters for silicon.

Plumbagin derived from the plant Plumbago indica, known as Chitrak in India, is an example of a medicinal compound used traditionally to cure a variety of ailments. Previous reports have indicated that it can inhibit the growth of Mycobacterium tuberculosis (Mtb), the causative agent of the deadly disease TB. In this investigation, we provide an insight into its mode of action. We show here that a significant mycobacterial target that is inhibited by plumbagin is the enzyme ThyX, a form of thymidylate synthase, that is responsible for the synthesis of dTMP from dUMP in various bacterial pathogens, including Mtb. Using a purified preparation of the recombinant version of Mtb ThyX, we demonstrate that plumbagin, a 2,4 napthoquinone, but not lawsone, a structurally related medicinal compound, inhibits its activity in vitro. We also show that the intracellular [dTTP]/[dATP] ratio in Mycobacterium smegmatis (Msm) cells decrease upon treatment with plumbagin, and this, in turn, leads to cell death. Such a conclusion is supported by the observation that over-expression of thyx in the plumbagin treated Msm cells leads to the restoration of viability. The results of our investigation indicate that plumbagin kills mycobacterial cells primarily by targeting ThyX, a vital enzyme required for their survival.

Aptamer based sensors have been observed to have excellent recognition capacity towards various types of target molecules such as metal ions, proteins and other biomolecules. In this study, we report an optical ‘turn off’ nanosensor, which consists of a deoxyribonucleic acid aptamer attached to a quantum dot on the 5’ terminus and gold nanoparticle on the 3’ terminus. The sensor uses the principle of fluorescence resonance energy transfer to detect C - reactive protein. With an increase in the target concentration in the sensor, the photoluminescence intensity decreases resulting in an increase in quenching efficiency. The nanosensor has a limit of detection as low as 1.77 picomolar and is observed to be highly specific to C-reactive protein. It also successfully detects the target protein in clinical samples exhibiting around 10% quenching at 10 picomolar target concentration. Future work on this sensor will involve investigation of this sensor in an in vivo environment so that it can be deployed for inflammatory disease management.

Cu2+ based distance measurements using the double-histidine (dHis) motif by pulsed ESR present an attractive strategy to obtain precise, narrow distance distributions that can be easily related to protein backbone structure (Cunningham et al., Angew. Chem., Int. Ed., 2015, 54, 633). The Cu2+-ion is introduced as a complex with the iminodiacetic acid (IDA) chelating agent, which enhances binding selectivity to the two histidine residues that are site-selectively placed on the protein through mutagenesis. However, initial results of this method produced weak dipolar modulations. To enhance applicability of the double histidine motif using IDA, we perform a systematic examination of the possible causes of these weak dipolar modulations. We examine the efficiency of the Cu2+-ion to form the Cu2+–IDA complex in solution. In addition, we analyze the selectivity of Cu2+–IDA binding to dHis sites at both α-helical and β-strand environments. Our results indicate that the dHis motif on the β-sheet sites have high affinity towards Cu2+–IDA while the dHis sites on α-helices show poor affinity for the metal-ion complex. We are able to use our new findings to optimize conditions to maximize dHis loading while minimizing both free Cu2+ and unbound Cu2+–IDA complex in solution, allowing us to double the sensitivity of the Double Electron–Electron Resonance (DEER) experiment. Finally, we illustrate how Cu2+-based CW-ESR and DEER can be combined to obtain information on populations of different Cu2+-complexes in solution.

Osteomyelitis, an infectious disease predominantly tied to poor sanitary conditions in underdeveloped regions of the world, is in need of inexpensive, easily in situ synthesizable and administrable materials for its treatment. The results of this study stem from the attempt to create one such affordable and minimally invasive therapeutic platform in the form of a self-setting, injectable cement with a tunable drug release profile, composed of only nanoparticulate hydroxyapatite, the synthetic version of the bone mineral. Cements comprised two separately synthesized hydroxyapatite powders, one of which, HAP2, was precipitated abruptly, retaining the amorphous nature longer, and the other one of which, HAP1, was precipitated at a slower rate, more rapidly transitioning to the crystalline structure. Cements were made with four different weight ratios of the two hydroxyapatite components: 100/ 0, 85/15, 50/50, and 0/100 with respect to HAP1 and HAP2. Both the setting and the release rates measured on two different antibiotics, vancomycin and ciprofloxacin, were controlled using the weight ratio of the two hydroxyapatite components. Various inorganic powder properties were formerly used to control drug release, but here we demonstrate for the first time that the kinetics of the mechanism of formation of a solid compound can be controlled to produce tunable drug release profiles. Specifically, it was found that the longer the precursor calcium phosphate component of the cement retains the amorphous nature of the primary precipitate, the more active it was in terms of speeding up the diffusional release of the adsorbed drug. The setting rate was, in contrast, inversely proportional to the release rate and to the content of this active hydroxyapatite component, HAP2. The empirical release profiles were fitted to a set of equations that could be used to tune the release rate to the therapeutic occasion. All of the cements loaded with vancomycin or ciprofloxacin inhibited the growth of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli and Pseudomonas aeruginosa in both agar diffusion assays and broth dilution tests with intensities either comparable to the antibiotic per se, as in the case of ciprofloxacin, or even larger than the antibiotic alone, as in the case of vancomycin. Interestingly, even the pure cements exhibited an antibacterial effect ranging from moderate to strong, while demonstrating high levels of biocompatibility with osteoclastic RAW264.7 cells and only slightly affecting the viability of the osteoblastic MC3T3-E1 cells, in direct proportion with the amount of the more active hydroxyapatite component in the cements. This antibacterial effect was especially noticeable against Gram-negative bacteria, where the growth inhibition by the cements was comparable to or even stronger than that of the pure antibiotics. The antibiofilm assay against P. aeruginosa biofilms reiterated the antibiotic effectiveness of pure, antibiotic-free cements. That the carrier per se, composed of a nontoxic, easily prepared, bone mineral composite, can exhibit a strong antibacterial effect even in the absence of an antibiotic drug is an insight highly relevant in view of the rising resistance of an array of pathogens to traditional antibiotic therapies and the demands for the timely development of suitable alternatives.

Endospore formation under environmental stress conditions is a well-established phenomenon for members of bacterial phylum Firmicutes, among which the most well studied ones belong to genus Bacillus and Clostridium. So far, known sizes of the spores are all larger than 500 nm. Nano-forms of bacteria have been reported but the notion still remains controversial.In this study, we provide visual evidences of living nano-entities (named here as ‘nano-spores’) formed by a bacterial species Bacillus cereus under prolonged stress, which are capable of escaping though standard sterile filtration procedure. The existence of nano-forms of bacteria was initially identified in a yeast ribosome preparation. We further demonstrate the transformation of the ‘nano-spores’ into mature cells upon nutrient supply. Our study not only demonstrates the ability of bacteria to get transformed into yet-unknown form in order to survive under harsh environment, but also brings to light the existence of the smallest possible form of life.

Bone grafting is one of the commonest surgical procedures, yet all bone substitutes developed so far suffer from specific weaknesses and the search for a bone graft material with ideal physical and biological properties is still ongoing. Calcium phosphate pastes are the most frequently used synthetic bone grafts, yet they (a) often take an impractically long time to set, (b) release the drug content too fast, and (c) do not form pores large enough to accommodate host cells and foster osseointegration. To make up for these deficiencies, we introduced gelatin and silica to pastes composed of 5–15 nm sized hydroxyapatite nanoparticles and yielded a bioresorbable composite that is compact, yet flowing upon injection; that prevents setting at room temperature, but sets promptly, in minutes, at 37 °C; that displays an increase in surface porosity following immersion in physiological fluids; that allows for sustained release of antibiotics; and that sets in a tunable manner and in clinically relevant time windows: 1–3 minutes at its fastest. Timelapse, in situ X-ray diffraction analysis demonstrated that the setting process is accompanied by an increase in crystallinity of the initially amorphous hydroxyapatite, involving no polymorphic phase transitions in its course. Setting time can be tuned by controlling the weight content of gelatin or powder-to-liquid ratio. The release of vancomycin was slow, ~ 8 % after 2 weeks, and unaffected by the gelatin content. While vancomycin-loaded pastes were effective in reducing the concentration of all bacterial species analyzed, the bacteriostatic effects of the antibiotic-free pastes were pronounced against S. liquefaciens and E. coli. S. liquefaciens bacilli underwent beading and filamentation during the treatment, suggesting that the antimicrobial effects are attributable to cell wall disruption by hydroxyapatite nanoparticles. Vancomycin-loaded pastes augmented the activity of the antibiotic against P. aeruginosa and S. liquefaciens, while exhibiting no negative effects against human mesenchymal stem cells. They were also uptaken three times more abundantly than pure hydroxyapatite, indicating the theoretical favorability of their use for intracellular delivery of therapeutics. This selectivity, toxic for bacteria and harmless for primary stem cells, is promising for application as bone grafts for osteomyelitis.

Electron paramagnetic resonance (EPR) has become an important tool to probe conformational changes in nucleic acids. An array of EPR labels for nucleic acids are available, but they often come at the cost of long tethers, are dependent on the presence of a particular nucleotide or can be placed only at the termini. Site directed incorporation of Cu2+-chelated to a ligand, 2,2′dipicolylamine (DPA) is potentially an attractive strategy for site-specific, nucleotide independent Cu2+-labelling in DNA. To fully understand the potential of this label, we undertook a systematic and detailed analysis of the Cu2+-DPA motif using EPR and molecular dynamics (MD) simulations. We used continuous wave EPR experiments to characterize Cu2+ binding to DPA as well as optimize Cu2+ loading conditions. We performed double electron-electron resonance (DEER) experiments at two frequencies to elucidate orientational selectivity effects. Furthermore, comparison of DEER and MD simulated distance distributions reveal a remarkable agreement in the most probable distances. The results illustrate the efficacy of the Cu2+-DPA in reporting on DNA backbone conformations for sufficiently long base pair separations. This labelling strategy can serve as an important tool for probing conformational changes in DNA upon interaction with other macromolecules.

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Despite the long history of nanoparticulate calcium phosphate (CaP) as a non-viral transfection agent, there has been limited success in attempts to optimize its properties for transfection comparable in efficiency to that of viral vectors. Here we focus on the optimization of: (a) CaP nanoparticle precipitation conditions, predominantly supersaturation and Ca/P molar ratios; (b) transfection conditions, mainly the concentrations of the carrier and plasmid DNA; (c) the presence of surface additives, including citrate anion and cationic poly(L-lysine) (PLL). CaP nanoparticles significantly improved transfection with plasmid DNA encoding enhanced green fluorescent protein (eGFP) in pre-osteoblastic MC3T3-E1 cells compared to a commercial non-viral carrier. At the same time they elicited significantly lesser cytotoxicity than the commercial carrier. Plasmid DNA acted as a nucleation promoter, decreasing the nucleation lag time of metastable CaP solutions and leading to a higher rate of nucleation and a lower size of the precipitated particles. The degree of supersaturation (DS) of 15 was found to be more optimal for transfection than that of 12.5 or 17.5 and higher. Because CaP particles precipitated at DS 15 were spherical, while DS 17.5 and 21 yielded acicular particles, it was concluded that spherical particle morphologies were more conducive to transfection than the anisotropic ones. Even though the yield at DS 15 was 10 and 100 times lower than that at DS 17.5 and 21, respectively, transfection rates were higher using CaP nanoparticle colloids prepared at DS 15 than using those made at higher or lower DS, indicating that the right particle morphology can outweigh the difference in the amount of the carrier, even when this difference is close to 100x. In contrast to the commercial carrier, the concentration of CaP-pDNA delivered to the cells was directly proportional to the transfection rate. Osteosarcoma K7M2 cells were four times more easily transfectable with CaP nanoparticles than the MC3T3-E1 cells. The addition of citrate increased the transfection rate at lower concentrations; however, a complete redispersal of CaP-pDNA nanoparticles at higher concentrations of citrate coincided with a complete diminishment of transfection, implying the benefits of partial aggregation of CaP nanoparticles carrying pDNA. In contrast, PLL delayed transfection initially, but enhanced it at longer time points (≥ 96 h), leading to the conclusion that both citrate and PLL could exert positive effects on transfection: citrate if added at low concentrations and PLL to extend transfection over longer periods of time.