A fast and simple FIA-chemiluminescence method for the evaluation of Roselle flowers as scavenger of the free radicals generated by UV irradiated antibiotics.

This work proposes a new method for the in vitro evaluation of the effect of UV irradiation on the production of free radicals and other reactive species during the photodecomposition of drugs. The method was based on the UV irradiation of antibiotics molecules to generate excited states that undergo to homolytic bond cleavages. These reactive species can be detected by their ability to oxidize the luminol, producing the electronically excited aminophtalate, which decays to the ground state releasing electromagnetic radiation in the visible zone of the spectrum. This method was applied to penicillin G, nafcillin, azlocillin and neomycin dissolved in water. It was found that the intensity of the luminol chemiluminescence emission (CL) was proportional to the concentration and dependent on the molecular structure of these drugs. Under the optimized conditions, it was found that penicillin and azlocillin were the most susceptible to photodegradation, while neomycin sulfate was the less affected by the UV light. It was observed that the addition to the antibiotics dissolutions of a hydro-alcoholic extract of petals of calyxes of Roselle reduced the CL intensity, indicating that the extract was able to scavenge the free radicals in the irradiated drugs. This result suggest that its addition to the antibiotics can help in the protection against the radicals formed during the exposition to solar light of patients treated with topic similar antibiotics.

Polyphenol-Assisted Exfoliation of Transition Metal Dichalcogenides into Nanosheets as Photothermal Nanocarriers for Enhanced Antibiofilm Activity.

Transition metal dichalcogenide (TMD) nanosheets have evoked enormous research enthusiasm and have shown increased potentials in the biomedical field. However, a great challenge lies in high-throughput, large-scale, and eco-friendly preparation of TMD nanosheet dispersions with high quality. Herein, we report a universal polyphenol-assisted strategy to facilely exfoliate various TMDs into monolayer or few-layer nanosheets. By optimizing the exfoliation condition of molybdenum disulfide (MoS2), the yield and concentration of as-exfoliated nanosheets are up to 60.5% and 1.21 mg/mL, respectively. This is the most efficient aqueous exfoliation method at present and is versatile for the choices of polyphenols and TMD nanomaterials. The as-exfoliated MoS2 nanosheets possess superior biomedical stability as nanocarriers to load antibiotic drugs. They show a high photothermal conversion effect and thus induce a synergetic effect of chemotherapy and photothermal therapy to harvest enhanced antibiofilm activity under near-infrared (NIR) light. All these results offer an appealing strategy toward the synthesis and application of ultrathin TMD nanosheets, with great implications for their development.

Modeling covalent-modifier drugs.

In this review, we present a summary of how computer modeling has been used in the development of covalent-modifier drugs. Covalent-modifier drugs bind by forming a chemical bond with their target. This covalent binding can improve the selectivity of the drug for a target with complementary reactivity and result in increased binding affinities due to the strength of the covalent bond formed. In some cases, this results in irreversible inhibition of the target, but some targeted covalent inhibitor (TCI) drugs bind covalently but reversibly. Computer modeling is widely used in drug discovery, but different computational methods must be used to model covalent modifiers because of the chemical bonds formed. Structural and bioinformatic analysis has identified sites of modification that could yield selectivity for a chosen target. Docking methods, which are used to rank binding poses of large sets of inhibitors, have been augmented to support the formation of protein-ligand bonds and are now capable of predicting the binding pose of covalent modifiers accurately. The pKa's of amino acids can be calculated in order to assess their reactivity towards electrophiles. QM/MM methods have been used to model the reaction mechanisms of covalent modification. The continued development of these tools will allow computation to aid in the development of new covalent-modifier drugs. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.

Inhalable Andrographolide-ß-cyclodextrin Inclusion Complexes for Treatment of Staphylococcus aureus Pneumonia by Regulating Immune Responses.

Bacterial pneumonia is a serious disease with high mortality if no appropriate and immediate therapy is available. Andrographolide (AG) is an anti-inflammatory agent extracted from a traditional Chinese herb andrographis paniculata. Oral AG tablets and pills are clinically applied for treatment of upper respiratory tract infections. However, the low solubility and bioavailability of AG lead to high doses and long-term therapy. Here we developed an andrographolide-ß-cyclodextrin inclusion complex (AG-ß-CD) for inhalation therapy of Staphylococcus aureus pneumonia. AG-ß-CD was identified with X-ray diffraction and FT-IR. Surprisingly, both AG-ß-CD and AG showed little in vitro anti-S. aureus activity. However, pulmonary delivery of AG, AG-ß-CD, or penicillin had significant anti-S. aureus pneumonia effects. Leukocytes, neutrophils, white blood cells, total proteins, TNF-α, IL-6, NF-κB p65 expression, and bacterial colonies in the bronchoalveolar lavage fluids were detected. Pulmonary delivery of AG and AG-ß-CD led to bacterial inhibition and inflammation alleviation by regulating immune responses, while penicillin only killed bacteria without significant immune regulation. Moreover, the antipneumonia activity of AG-ß-CD was much higher than that of AG, probably resulting from locally accelerated AG dissolution due to ß-CD inclusion. The aerodynamic diameter of AG-ß-CD powders was 2.03 µm, suitable for pulmonary delivery. Inhalable AG-ß-CD is a promising antibacterial and anti-inflammatory medicine for the treatment of S. aureus pneumonia by regulating immune responses, and the effect is enhanced by ß-CD inclusion. AG and its formulations might be potent weapons against the resistant bacterial pneumonia due to their specific mechanism in the future.

Direct, rapid, and label-free detection of enzyme-substrate interactions in physiological buffers using CMOS-compatible nanoribbon sensors.

We demonstrate the versatility of Al2O3-passivated Si nanowire devices ("nanoribbons") in the analysis of enzyme-substrate interactions via the monitoring of pH change. Our approach is shown to be effective through the detection of urea in phosphate buffered saline (PBS), and penicillinase in PBS and urine, at limits of detection of <200 µM and 0.02 units/mL, respectively. The ability to extract accurate enzyme kinetics and the Michaelis-Menten constant (Km) from the acetylcholine-acetylcholinesterase reaction is also demonstrated.

Structure-selective hot-spot Raman enhancement for direct identification and detection of trace penicilloic acid allergen in penicillin.

Trace penicilloic acid allergen frequently leads to various fatal immune responses to many patients, but it is still a challenge to directly discriminate and detect its residue in penicillin by a chemosensing way. Here, we report that silver-coated gold nanoparticles (Au@Ag NPs) exhibit a structure-selective hot-spot Raman enhancement capability for direct identification and detection of trace penicilloic acid in penicillin. It has been demonstrated that penicilloic acid can very easily link Au@Ag NPs together by its two carboxyl groups, locating itself spontaneously at the interparticle of Au@Ag NPs to form strong Raman hot-spot. At the critical concentration inducing the nanoparticle aggregation, Raman-enhanced effect of penicilloic acid is ~60,000 folds higher than that of penicillin. In particular, the selective Raman enhancement to the two carboxyl groups makes the peak of carboxyl group at C6 of penicilloic acid appear as a new Raman signal due to the opening of ß-lactam ring of penicillin. The surface-enhanced Raman scattering (SERS) nanoparticle sensor reaches a sensitive limit lower than the prescribed 1.0‰ penicilloic acid residue in penicillin. The novel strategy to examine allergen is more rapid, convenient and inexpensive than the conventional separation-based assay methods.

Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli.

BACKGROUND: Penicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of ß-lactam antibiotics. Development of a PAC with a longer half-life under the reaction conditions used is essential for the improvement of the operational stability of the process. A gene encoding a homologue to Escherichia coli PAC was found in the genome of the thermophilic bacterium Thermus thermophilus (Tth) HB27. Because of the nature of this PAC and its complex maturation that is crucial to reach its functional heterodimeric final conformation, the overexpression of this enzyme in a heterologous mesophilic host was a challenge. Here we describe the purification and characterization of the PAC protein from Tth HB27 overexpressed in Escherichia coli. RESULTS: Fusions to a superfolder green fluorescent protein and differential membrane solubilization assays indicated that the native enzyme remains attached through its amino-terminal end to the outer side of the cytoplasmic membrane of Tth cells. In order to overexpress this PAC in E. coli cells, a variant of the protein devoid of its membrane anchoring segment was constructed. The effect of the co-expression of chaperones and calcium supplementation of the culture medium was investigated. The total production of PAC was enhanced by the presence of DnaK/J and GrpE and even more by trigger factor and GroEL/ES. In addition, 10 mM calcium markedly improved both PAC specific and volumetric activities. Recombinant PAC was affinity-purified and proper maturation of the protein was confirmed by SDS-PAGE and MALDI-TOF analysis of the subunits. The recombinant protein was tested for activity towards several penicillins, cephalosporins and homoserine lactones. Hydrophobic acyl-chain penicillins were preferred over the rest of the substrates. Penicillin K (octanoyl penicillin) was the best substrate, with the highest specificity constant value (16.12 mM-1.seg-1). The optimum pH was aprox. 4 and the optimum temperature was 75 °C. The half-life of the enzyme at this temperature was 9.2 h. CONCLUSIONS: This is the first report concerning the heterologous expression of a pac gene from a thermophilic microorganism in the mesophilic host E. coli. The recombinant protein was identified as a penicillin K-deacylating thermozyme.

Self-assembled squalenoylated penicillin bioconjugates: an original approach for the treatment of intracellular infections.

We describe here new nanoparticles based on the bioconjugation of penicillin G to squalene in order to overcome severe intracellular infections by pathogen bacteria whose mechanism of resistance arises from the poor intracellular diffusion of several antibiotics. Two different squalene-penicillin G conjugates were synthesized (pH-sensitive and pH-insensitive), and their self-assembly as nanoparticles was investigated through morphology and stability studies. These nanoparticles had a size of 140 ± 10 nm (polydispersity index of 0.1) and a negative charge, and they did not display any supramolecular organization. Furthermore, they were found stable in water and in different culture medium. The cellular uptake and localization of these fluorescently labeled nanoparticles were explored on the macrophage cell line J774 by flow cytometry and confocal microscopy analysis. The squalenoylated nanoparticles were found to be cell internalized through clathrin-dependent and -independent endocytic pathways. Moreover, they induced an improved intracellular antibacterial activity on the facultative intracellular pathogen S. aureus, compared with free penicillin G, despite the absence of co-localization between the bacteria and the nanoparticles in the cells. This study suggests that the bioconjugation of an antibiotic to a squalene template could be a valuable approach for overcoming the antibiotic resistance due to intracellular bacterial infections.

Effects of different calcium, magnesium, and zinc concentrations supplemented on hepatopancreatic cell proliferation of kuruma prawn, Penaeus japonicus.

The effects of different calcium (Ca(2+)), magnesium (Mg(2+)), and zinc (Zn(2+)) concentrations supplemented on hepatopancreatic cell proliferation of kuruma prawn, Penaeus japonicus was studied. The culture system consists of medium 199 (M 199) supplemented with 0.060 mol/L NaCl, 1.011 g/L glucose, 1,000 UI/ml penicillin, 1,000 µg/ml treptomycin, heat inactivated fetal calf serum (FCS) 20% for primary cell culture and 10% for subculture. The RNA/DNA ratio of the cell cultures was measured. The results show that the cell division of hepatopancreatic cells of P. japonicus was enhanced by the optimal concentration of inorganic salt (Ca(2+), 1.0 g/L; Mg(2+), 5.0 g/L; Zn(2+), 80 µg/L). The hepatopancreatic cell culture system and improved culture conditions described here will be very useful for in vitro experiments to study viruses responsible for infections in shrimp leading to tremendous economic losses.

Hypersensitivity reactions to penicillins: studies in a group of patients with negative benzylpenicillin G skin test.

BACKGROUND: Although skin tests are usually employed to evaluate current penicillin allergy status, a negative result does not exclude hypersensitivity. There is a need for accurate in vitro tests to exclude hypersensitivity. A radioallergosorbent test (RAST) is a potentially good supplementary approach, but there is little information on the suitability of this method to diagnose penicillin hypersensitivity in subjects with a negative skin test to benzylpenicillin. METHODS: A total of 133 patients with a negative skin test to benzylpenicillin G (PG) and all of whom developed allergic reactions to PG were studied. RAST was used to detect eight kinds of specific IgE antibodies to penicillins in serum, which included four kinds of major and minor antigenic determinants to four penicillin drugs. The combination sites for the specific IgE antibodies were studied by RAST inhibition test. RESULTS: The rate of positive reactions for the specific IgE antibodies was 59.40% (79/133). Of the eight kinds of antigenic determinants, the positive rates for specific IgE against the major and minor determinants were 39.10% (52) and 42.86% (57) respectively. Of the four drugs, positive cases only to PG were 10 (7.5%), were significantly fewer than the cross-reacting positive cases (36) to PG (P < 0.01). In the RAST inhibition studies all drugs exhibited good inhibitory potencies, and in some instances the side-chain of the penicillins could induce specific responses with a variable degree of cross-reactivity among the different penicillins. CONCLUSION: Radioallergosorbent test is a good complementary test in persons who are skin-test negative with PG, and the sensitivity of RAST increases with increasing specificity of IgE antibodies to be detected. 6-APA and the groups, making part of the different side-chains on penicillins, all contributed to the cross-reactivity.

Redefining penems.

The antimicrobial class of penems has the potential to address most of the relevant resistance issues associated with beta-lactam antibiotics because of their exceptionally broad spectrum of antibacterial activity and their intrinsic stability against hydrolytic attack by many beta-lactamases including ESBL and AmpC enzymes. The subclass of carbapenems covers the spectrum of hospital pathogens whereas the subclass of penems covers community pathogens. The only currently available penem, faropenem, has a low propensity for resistance development, beta-lactamase induction and selection of carbapenem-resistant Pseudomonas aeruginosa. This makes it attractive for the treatment of community-acquired infections and for step-down or sequential therapy following carbapenem treatment without jeopardizing the activity of carbapenems or the entire beta-lactam class in the hospital environment.

Diversity in domain architectures of Ser/Thr kinases and their homologues in prokaryotes.

BACKGROUND: Ser/Thr/Tyr kinases (STYKs) commonly found in eukaryotes have been recently reported in many bacterial species. Recent studies elucidating their cellular functions have established their roles in bacterial growth and development. However functions of a large number of bacterial STYKs still remain elusive. The organisation of domains in a large dataset of bacterial STYKs has been investigated here in order to recognise variety in domain combinations which determine functions of bacterial STYKs. RESULTS: Using sensitive sequence and profile search methods, domain organisation of over 600 STYKs from 125 prokaryotic genomes have been examined. Kinase catalytic domains of STYKs tethered to a wide range of enzymatic domains such as phosphatases, HSP70, peptidyl prolyl isomerases, pectin esterases and glycoproteases have been identified. Such distinct preferences for domain combinations are not known to be present in either the Histidine kinase or the eukaryotic STYK families. Domain organisation of STYKs specific to certain groups of bacteria has also been noted in the current anlaysis. For example, Hydrophobin like domains in Mycobacterial STYK and penicillin binding domains in few STYKs of Gram-positive organisms and FHA domains in cyanobacterial STYKs. Homologues of characterised substrates of prokaryotic STYKs have also been identified. CONCLUSION: The domains and domain architectures of most of the bacterial STYKs identified are very different from the known domain organisation in STYKs of eukaryotes. This observation highlights distinct biological roles of bacterial STYKs compared to eukaryotic STYKs. Bacterial STYKs reveal high diversity in domain organisation. Some of the modular organisations conserved across diverse bacterial species suggests their central role in bacterial physiology. Unique domain architectures of few other groups of STYKs reveal recruitment of functions specific to the species.

Microcalorimetric evaluation of the in vitro compatibility of amoxicillin/clavulanic acid and ampicillin/sulbactam with ciprofloxacin.

Solution calorimetric technique has been used to determine the compatibility of binary and ternary systems of ampicillin trihydrate (AMP), sulbactam sodium (SS), amoxicillin trihydrate (AM), potassium clavulanate (PC) and ciprofloxacin hydrochloride (CP). The enthalpy of solution (DeltasolH) were obtained over a wide range of composition in the pH range 2-9. For all the pure drugs the DeltasolH is endothermic in nature. The molar enthalpies of interaction of binary (DeltaHbi.E) and ternary (DeltaHter.E) mixtures of the drugs in aqueous buffers have been determined. The DeltaHbi.E for all binary systems is negative and pH dependent (maximum pH 6-8) indicating the interaction among charged species of the drugs. In case of binary systems with CP the magnitude of DeltaHbi.E indicate strong interactions. The variation and magnitude of DeltaHbi.E for the systems is discussed in terms of hydrogen bonding and van der Waal's interaction in the solution. The interaction parameter for ternary systems (A) is positive indicating repulsive interaction among the drugs. The coefficients hi's calculated from Redlich-Kister equation for binary systems (DeltaHbi.E) and ternary interaction parameter (A) were used to predict the compatibility of the marketed formulations in pH range studied.

beta-Lactam drug allergens: fine structural recognition patterns of cephalosporin-reactive IgE antibodies.

Lack of experimental findings on the spectrum of cephalosporin allergenic determinants has hindered diagnosis of adverse reactions to these drugs and retarded understanding of allergenic cross-reactions between cephalosporins and between cephalosporins and penicillins. Subjects allergic to the widely used cephalosporin antibiotic cefaclor have serum immuno globulin (Ig) E antibodies that react with the drug. Quantitative hapten inhibition studies employing sera from subjects allergic to cefaclor revealed fine structural recognition differences between the combining site specificities of cefaclor-reactive IgE antibodies in the sera of different subjects. Unlike penicillins, where discrete side chain or thiazolidine ring determinants alone may be recognized, IgE binding determinants on cefaclor encompassed the entire molecule. Fine structural recognition specificity differences at positions R1 (side-chain) and R2 (substituent attached to dihydrothiazine ring) were detected between IgE antibodies in different sera. Some antibodies showed clear preferential recognition of the aminobenzyl group at position R1 and Cl at R2 while with others, a greater degree of recognition tolerance was seen at R1 where, for example, the aminohydroxybenzyl or aminodihydrobenzyl groups were recognized, and at R2 where a methyl or even an ester group was tolerated. As with the penicillins, cephalosporins as allergens cannot simply be considered as a group of compounds with a common allergenic determinant structure. IgE antibodies that bind to cefaclor show great heterogeneity indicated by clear, fine structural differences in recognition of the R1 and R2 groups on the drug.