Fabrication of gelatin/silk fibroin/phage nanofiber scaffold effective against multidrug resistant Pseudomonas aeruginosa.

OBJECTIVE: The alarming rise of multi-drug resistant (MDR) Pseudomonas aeruginosa has prompted the World Health Organization to consider it a serious threat to human health. Although phage (Phg), an effective antibacterial treatment option, can maintain long-term infectivity via lyophilized storage, freeze-drying can be expensive and time-consuming. Thus, we propose electrospun gelatin/fibroin (G/F) nanofibrous formulation for dehydrating and storing phage against MDR P. aeruginosa. SIGNIFICANCE: The formulation of phage within the nanofibrous structure of the electrospun G/F scaffold would result in antimicrobial activity against MDR P. aeruginosa leading to enhanced wound healing. METHODS: Phg effective against MDR P. aeruginosa was isolated, characterized and loaded within G/F nanofibers by electrospinning. Morphology, crystallinity and thermal stability as well as the antimicrobial activity and the biocompatibility of the developed G/F/Phg nanofibers were determined. RESULTS: Phg-loaded G/F nanofibers revealed an amorphous structure with good thermal stability at temperatures below 300 °C and exhibited effective antibacterial activity against MDR P. aeruginosa with ∼2 log reduction in the bacterial count which increased to ∼4 log reduction in bacterial count after 16 h as compared to both the G/F nanofibers and the negative control. Lack of cytotoxic effects on cultured fibroblasts supported the biocompatibility of G/F/Phg nanofibers. CONCLUSION: The developed G/F/Phg nanofibers are able to maintain the viability of phage and represent a promising antimicrobial dressing for wounds infected with MDR P. aeruginosa.

A sensitive colorimetric assay for identification of Acinetobacter baumannii using unmodified gold nanoparticles.

AIMS: Acinetobacter baumannii is a global health problem, which threatens many healthcare settings. The current study aims to develop a detection assay for Ac. baumannii using unmodified gold nanoparticles (AuNPs). METHODS AND RESULTS: Fifty-three Ac. baumannii clinical isolates were collected from Egyptian hospitals. Bacterial isolation and biochemical identification of isolates were carried out followed by DNA extraction using boiling method and PCR amplification of the 23S-16S rRNA intergenic spacer sequences (ITS). AuNPs were synthesized using citrate reduction method. Detection and optimization of Ac. baumannii amplicons using unmodified spherical AuNPs were performed using species-specific DNA oligonucleotide. The nano-gold assay was able to colorimetrically detect and distinguish Ac. baumannii from other Gram-negative bacteria. The turnaround time of the assay is about 2 h including sample treatment and amplification. The assay detection limit is 0·8125 ng of DNA. CONCLUSIONS: The developed colorimetric assay is sensitive, fast and reliable and can be used for identification of Ac. baumannii. SIGNIFICANCE AND IMPACT OF THE STUDY: There is a need to develop robust, rapid, and specific methods for detection of Ac. baumannii isolated from clinical specimens. The developed nanogold assay prototype allows sensitive, specific and rapid detection of amplified DNA of A. baumannii and represents a reliable diagnostic tool to aid routine laboratory identification of this pathogen.

Characteristics of myoglobin, carbonic anhydrase III and the myoglobin/carbonic anhydrase III ratio in trauma, exercise, and myocardial infarction patients.

Carbonic anhydrase III (CA III) is an enzyme present in skeletal muscle which is released into circulation following injury. Myoglobin (Mb) is a heme protein located in skeletal, smooth, and cardiac muscle which is also released after injury. Because CA III is not present in myocardium, combining serum CA III and Mb measurements may improve the specificity of Mb as an early diagnostic marker for myocardial infarction (MI) provided that a fixed ratio of Mb and CA III is released from skeletal muscle following cell injury. We examined release of Mb and CA III for exercise subjects (n=12), trauma patients (n=18), and MI patients (n=10) following emergency department admission. A fixed ratio of Mb/CA III had medians of 3.505 (range: 1.05-6.76) and 2.890 (range: 0.97-3.97) for exercise and trauma subjects, respectively, in samples collected within 5 h of the event. The Mb/CA III ratio was significantly higher (P<0.001) in MI patients (median: 35.395; range: 8.65-170.45) during this same time. This study confirmed that Mb and CA III are released in a fixed ratio following exercise, showed no significant difference in the ratio for trauma patients, and demonstrated significant ratio elevation for MI patients. These data suggest the ratio to be a useful diagnostic indicator of MI.

Standardization of creatine kinase-MB (CK-MB) mass assays: the use of recombinant CK-MB as a reference material.

BACKGROUND: The AACC assembled a committee to identify and validate a standard creatine kinase MB isoenzyme (CK-MB) material to improve the comparability of CK-MB mass assays. METHODS: Three protocols were used. In protocol I, various CK-MB materials prepared in different matrices were screened as candidate standards. In protocol II, participating manufacturers calibrated their systems with concentrates of human heart CK-MB and then tested 20 patient samples to evaluate calibration bias. In protocol III, participating manufacturers calibrated their immunoassay systems using recombinant CK-MB2 (rCK-MB2) diluted into their respective sample diluents and measured 50 samples. RESULTS: Candidate materials showed high recovery in stripped human serum, but bias improved only from 59% to 38%. These data led to the use of human heart CK-MB diluted in each manufacturer's sample diluent. This strategy reduced bias from 31% to 15%. Because human heart CK-MB is difficult to provide, a lyophilized source of CK-MB2 was identified. rCK-MB2 was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reversed-phase HPLC, intrinsic protein fluorescence, circular dichroism, agarose gel electrophoresis, immunoreactivity studies, high and low temperature stability, and reconstituted stability to be equivalent to human heart CK-MB. Calibration of immunoassay systems with rCK-MB2 added into each respective manufacturer's sample diluent showed a 13% between-manufacturer bias. CONCLUSION: Lyophilized rCK-MB2 was determined suitable for use as a reference material for CK-MB mass assays.

Multisite study of a second generation whole blood rapid assay for cardiac troponin T.

We evaluated a second generation, qualitative, whole blood rapid assay for cardiac troponin T (cTnT), which employs a more cardio-specific troponin T mouse monoclonal capture antibody. Using quantitative cTnT enzyme-linked immunosorbent assay (ELISA) results as the benchmark for accuracy, we compared the performance of the second generation and the original whole blood rapid assays in 445 samples from patients with the following diagnoses, determined by medical record review: myocardial infarction, coronary bypass surgery, ischaemic heart disease, musculoskeletal disease, renal failure or other noncardiac conditions. Overall, concordance between the second generation cTnT Rapid Assay and the quantitative cTnT ELISA, compared using the McNemar test and a cut-off concentration of 0.1 microgram/L, was in the range 76-94% for each patient group. Using a receiver operating characteristic plot, the cut-off for the second generation cTnT Rapid Assay was in the range 0.06-0.08 microgram/L. We conclude that the second generation cTnT whole blood assay has a 2.5-fold lower analytical cut-off than the original rapid assay (0.2 microgram/L) and may represent a more sensitive clinical tool for the rapid triage and risk stratification of cardiac patients.

Biochemical markers of the acute coronary syndromes.

The acute coronary syndromes represent a continuum of myocardial ischemia ranging from angina, reversible tissue injury --> unstable angina, frequently associated with minor myocardial damage --> myocardial infarction and extensive tissue necrosis. Historically, coronary artery disease assessment has been mainly binary, using WHO criteria of symptoms, electrocardiography, and biochemical markers. The creatine kinase-MB isoenzyme (CK-MB) has been a benchmark for markers, but it is not specific for myocardium. Cardiac-specific isoforms of troponin T and I have emerged as sensitive myocardial infarction (MI) indicators and, importantly, for risk stratification of acute coronary syndrome patients. In addition to markers of myocardial cell necrosis, markers of plaque disruption (C-reactive protein and serum amyloid A), "angry" platelets (P-selectin), ischemia (glycogen phosphorylase-BB isoenzyme), and the procoagulant state and thrombosis (soluble fibrin) have potential use. Also, CK-MB and myoglobin have been combined with clinical indicators for monitoring reperfusion after thrombolytic therapy. Biochemical markers will continue to be an important clinical adjunct for MI diagnosis, risk assessment, and reperfusion monitoring in the future.

Abbott AxSYM Vancomycin II assay: multicenter evaluation and interference studies.

The authors evaluated the performance characteristics of the Abbott AxSYM Vancomycin II immunoassay in sera of patients with (n = 93 samples) and without (n = 327 patients) renal dysfunction. Correlation of vancomycin measurements with the Abbott AxSYM Vancomycin, Abbott TDx/TDxFLx, Syva enzyme-multiplied immunoassay technique (EMIT), DuPont automated chemistry analyzer (ACA), and high-performance liquid chromatography methods showed acceptable correlation as indicated by: slope values >0.95, r-values >0.97, y-intercepts <1.7 microg/ml, and S(y/x) ranging from 9% to 15% of the average vancomycin value. The AxSYM Vancomycin II assay showed acceptable correlation with AxSYM vancomycin, TDx/TDxFLx, and high-performance liquid chromatography methods in 93 samples from patients with renal dysfunction. This monoclonal antibody-based assay showed no apparent interference from the presence of human antimouse antibody (HAMA) or the microbiologically inactive vancomycin crystalline degradation product (CDP). The authors conclude that the AxSYM Vancomycin II assay showed satisfactory agreement with other methods tested in this study.

Production and characterization of monoclonal antibodies to human carbonic anhydrase III.

Carbonic anhydrase III (CAIII) is a cytosolic protein found almost exclusively in slow-oxidative skeletal muscle fibers. Upon excessive skeletal muscle activity or damage, CAIII is rapidly released into serum. CAIII is not found in cardiac muscle, whereas the muscle protein myoglobin (Myo) is found in skeletal and cardiac muscle. Because CAIII and Myo are released from injured muscle in a constant ratio, an increase in the Myo/CAIII ratio may be useful as an early diagnostic indicator of acute myocardial damage. Although several reliable Myo immunoassays have been established, no similar CAIII immunoassay is commercially available. We produced murine monoclonal antibodies (MAbs) to human CAIII using standard immunization and cell fusion procedures. Using an enzyme-linked immunoadsorbent assay (ELISA), three MAbs showed strong immunoreactivity with CAIII, but low to moderate levels of cross-reactivity with closely related isoenzymes CAI and CAII. The three MAbs demonstrated unique patterns of reactivity toward CAI, CAII, and CAIII, suggesting that different CAIII epitopes are recognized by the three MAbs. Specificity was further examined by Western blot analysis. These MAbs demonstrated potential for use in the development of an immunoassay for CAIII, and for investigating the biology of skeletal muscle injury in vivo.

Multicenter study of Abbott AxSYM Digoxin II assay and comparison with 6 methods for susceptibility to digoxin-like immunoreactive factors.

Performance characteristics of the Abbott nonpretreatment AxSYM Digoxin II assay were evaluated for quantification of digoxin at four independent sites. Correlation of digoxin measurements with the Abbott pretreatment AxSYM, Baxter Stratus II, Abbott TDx/ TDxFLx II, Abbott IMx, Emit 2000, and Beckman Synchron CX digoxin assays showed acceptable agreement, as indicated by: slope values > 0.84, r > 0.90, y-intercepts for all comparisons at or below the assay detection limit, and Sy/x ranging between 7.5% and 15.4% of the average digoxin value. Susceptibility to interference from digoxin-like immunoreactive factors (DLIFs) was examined in 233 samples from renal patients, liver disease patients, cord blood, and third-trimester pregnancies; the AxSYM Digoxin II assay demonstrated the least DLIFs interference. DLIF susceptibility for four of the methods was significantly greater (P < 0.05) than in the AxSYM Digoxin II assay; susceptibilities of the Stratus II and Emit 2000 methods were similar to the AxSYM Digoxin II assay.

Interaction of cationic liposomes with cells of electrically active neuronal networks in culture.

Incubation of rhodamine-labeled cationic liposomes with mature murine spinal cultures results in strong fluorescence that is evenly distributed on somata and neurites of neurons in 7 different cultures. Staining of the glial carpet is minimal. Rhodamine-labeled dextran, encapsulated in liposomes, also stains neurons. Electron microscope data show external attachment and intact internalization of liposomes. Spontaneous electrical bursting activity is altered but not lost after incubation.

The use of neuronal networks on multielectrode arrays as biosensors.

Mammalian spinal neuronal networks growing on arrays of photoetched electrodes in culture provide a highly stable system for the long-term monitoring of multichannel, spontaneous or evoked electrophysiological activity. In the absence of the homeostatic control mechanisms of the central nervous system, these networks show remarkable sensitivities to minute chemical changes and mimic some of the properties of sensory tissue. These sensitivities could be enhanced by receptor up-regulation and altered by the expression of unique receptors. The fault-tolerant spontaneous network activity is used as a dynamic platform on which large changes in activity signify detection of chemical substances. We present strategies for the expression of novel supersensitivities to foreign molecules via genetic engineering that involves the grafting of ligand binding cDNA onto truncated native receptor DNA and the subsequent expression of such chimeric receptors.

Production and characterization of antibodies against C-terminal peptide of protein F1: a novel phosphorylation at serine 209 of the peptide by protein kinase C.

Protein F1 (GAP-43, B-50, neuromodulin, P-57), a neural tissue-specific phosphoprotein enriched in the growth cones of elongating neurites, is suggested to be involved in synaptic plasticity, neuronal development, and neurotransmitter release. In this study, a 21 amino acid polypeptide (AKPKES*ARQDEGKEDPEADQE) that corresponds to the C-terminus sequence of protein F1 (from position 204-224) was synthesized and used to produce anti-protein F1 antibodies. Immunoblot analysis has demonstrated that the prepared antibodies recognized intact protein F1. Protein F1 and the synthesized F1 peptide were phosphorylated in vitro by PKC. Furthermore, phosphorylated protein F1 was immunoprecipitated by anti-F1 peptide antibodies demonstrating that these antibodies recognized both native, non-phosphorylated and phosphorylated protein. The anti-protein F1 antibodies also stained the plasma membranes of cell bodies and neuritis of mouse neuronal cultures obtained from 14-day old spinal embryonic tissue. By contrast, no glial cells were stained. These data suggest that serine 209 at the C-terminus of protein F1 may be a substrate for PKC phosphorylation in vivo. In addition, antibodies raised against F1 peptide revealed protein F1 immunoreactivity that outlined all neurites of cultured mouse spinal neurons.