Enhanced selectivity of hydrogel-based molecularly imprinted polymers (HydroMIPs) following buffer conditioning.

We have investigated the effect of buffer solution composition and pH during the preparation, washing and re-loading phases within a family of acrylamide-based molecularly imprinted polymers (MIPs) for bovine haemoglobin (BHb), equine myoglobin (EMb) and bovine catalyse (BCat). We investigated water, phosphate buffer saline (PBS), tris(hydroxymethyl)aminomethane (Tris) buffer and succinate buffer. Throughout the study MIP selectivity was highest for acrylamide, followed by N-hydroxymethylacrylamide, and then N-iso-propylacrylamide MIPs. The selectivity of the MIPs when compared with the NIPs decreased depending on the buffer conditions and pH in the order of Tris>PBS>succinate. The Tris buffer provided optimum imprinting conditions at 50mM and pH 7.4, and MIP selectivities for the imprinting of BHb in polyacrylamide increased from an initial 8:1 to a 128:1 ratio. It was noted that the buffer conditions for the re-loading stage was important for determining MIP selectivity and the buffer conditions for the preparation stage was found to be less critical. We demonstrated that once MIPs are conditioned using Tris or PBS buffers (pH7.4) protein reloading in water should be avoided as negative effects on the MIP's imprinting capability results in low selectivities of 0.8:1. Furthermore, acidifying the pH of the buffer solution below pH 5.9 also has a negative impact on MIP selectivity especially for proteins with high isoelectric points. These buffer conditioning effects have also been successfully demonstrated in terms of MIP efficiency in real biological samples, namely plasma and serum

Species distribution and antimicrobial susceptibility of gram-negative aerobic bacteria in hospitalized cancer patients

<p>Abstract</p> <p>Background</p> <p>Nosocomial infections pose significant threats to hospitalized patients, especially the immunocompromised ones, such as cancer patients.</p> <p>Methods</p> <p>This study examined the microbial spectrum of gram-negative bacteria in various infection sites in patients with leukemia and solid tumors. The antimicrobial resistance patterns of the isolated bacteria were studied.</p> <p>Results</p> <p>The most frequently isolated gram-negative bacteria were <it>Klebsiella pneumonia </it>(31.2%) followed by <it>Escherichia coli </it>(22.2%). We report the isolation and identification of a number of less-frequent gram negative bacteria (<it>Chromobacterium violacum</it>, <it>Burkholderia cepacia, Kluyvera ascorbata, Stenotrophomonas maltophilia, Yersinia pseudotuberculosis</it>, and <it>Salmonella arizona</it>). Most of the gram-negative isolates from Respiratory Tract Infections (RTI), Gastro-intestinal Tract Infections (GITI), Urinary Tract Infections (UTI), and Bloodstream Infections (BSI) were obtained from leukemic patients. All gram-negative isolates from Skin Infections (SI) were obtained from solid-tumor patients. In both leukemic and solid-tumor patients, gram-negative bacteria causing UTI were mainly <it>Escherichia coli </it>and <it>Klebsiella pneumoniae</it>, while gram-negative bacteria causing RTI were mainly <it>Klebsiella pneumoniae</it>. <it>Escherichia coli </it>was the main gram-negative pathogen causing BSI in solid-tumor patients and GITI in leukemic patients. Isolates of <it>Escherichia coli, Klebsiella, Enterobacter</it>, <it>Pseudomonas, and Acinetobacter </it>species were resistant to most antibiotics tested. There was significant imipenem -resistance in <it>Acinetobacter </it>(40.9%), <it>Pseudomonas </it>(40%), and <it>Enterobacter </it>(22.2%) species, and noticeable imipinem-resistance in <it>Klebsiell</it>a (13.9%) and <it>Escherichia coli </it>(8%).</p> <p>Conclusion</p> <p>This is the first study to report the evolution of imipenem-resistant gram-negative strains in Egypt. Mortality rates were higher in cancer patients with nosocomial <it>Pseudomonas </it>infections than any other bacterial infections. Policies restricting antibiotic consumption should be implemented to avoid the evolution of newer generations of antibiotic resistant-pathogens.</p

Dysregulation of micro-RNA contributes to the risk of unexplained recurrent pregnancy loss

Although there are plenty of evidence that dysregulation of microRNA (miRNA) level is involved in many human diseases, it is still unknown whether abnormal levels of specific miRNAs are associated with recurrent pregnancy loss (RPL). We believe that such an association do exist as this study confirmed significant differences in the level of specific miRNAs between RPL cases and healthy controls. The study was conducted on 100 Palestinian women: 60 patients with at least two unexplained consecutive pregnancy losses half of them were pregnant at the first trimester and the rest were non-pregnant and 40 healthy controls with at least two live births and no history of pregnancy loss; half of them were at their first trimester of pregnancy and the rest were non-pregnant. We investigated the relative expression of miR-21, miR-126, miR-155, miR-182, miR-222 and miR-517* using quantitative real-time polymerase chain reaction and Ct method experiments. Differential expression was evaluated using Student t-test and fold change analyses. The expression difference of miR-21, miR-126 and miR-182 between patients and controls in the pregnant subjects showed statistically significant difference (p-value ≤ 0.05) with fold decrease of 1.5, 1.6 and 5.6, respectively. In the non-pregnant group miR-21, miR-126, miR-222 and miR-517* expressions were significantly different with fold decrease of 2.4, 2.9, 2.7 and 11.8, respectively. In conclusion, at least miR-21 and miR-126 could serve as potential markers for idiopathic RPL as their levels were significantly lower in patients before being pregnant and during pregnancy. Moreover, restoration of the normal level of those miRNAs might be a novel intervention strategy in unexplained RPL

Determination of protein binding affinities within hydrogel-based molecularly imprinted polymers (HydroMIPs)

Hydrogel-based molecularly imprinted polymers (HydroMIPs) were prepared for several proteins (haemoglobin, myoglobin and catalase) using a family of acrylamide-based monomers. Protein affinity towards the HydroMIPs was investigated under equilibrium conditions and over a range of concentrations using specific binding with Hill slope saturation profiles. We report HydroMIP binding affinities, in terms of equilibrium dissociation constants (Kd) within the micro-molar range (25 ± 4 mM, 44 ± 3 mM, 17 ± 2 mM for haemoglobin, myoglobin and catalase respectively within a polyacrylamide-based MIP). The extent of non-specific binding or cross-selectivity for non-target proteins has also been assessed. It is concluded that both selectivity and affinity for both cognate and non-cognate proteins towards the MIPs were dependent on the concentration and the complementarity of their structures and size. This is tentatively attributed to the formation of protein complexes during both the polymerisation and rebinding stages at high protein concentrations. We have used atomic force spectroscopy to characterize molecular interactions in the MIP cavities using protein-modified AFM tips. Attractive and repulsive force curves were obtained for the MIP and NIP (non-imprinted polymer) surfaces (under protein loaded or unloaded states). Our force data suggest that we have produced selective cavities for the template protein in the MIPs and we have been able to quantify the extent of non-specific protein binding on, for example, a non-imprinted polymer (NIP) control surface

Spectroscopic and quartz crystal microbalance (QCM) characterisation of protein-based MIPs

We have studied acrylamide-based polymers of varying hydrophobicity (acrylamide, AA; N-hydroxymethylacrylamide, NHMA; N-isopropylacrylamide, NiPAm) for their capability of imprinting protein. Rebinding capacities (Q) from spectroscopic studies were highest for bovine haemoglobin (BHb) MIPs based on AA, Q = 4.8 ± 0.21 76 ± 0.5%). When applied to the QCM sensor as thin-film MIPs, NHMA MIPs were found to exhibit best discrimination between MIP and non-imprinted control polymer (NIP) in the order of NiPAm < AA < NHMA. The extent of template removal and rebinding, using both crystal impedance and frequency measurements, demonstrated that 10% (w/v):10% (v/v) sodium dodecyl sulphate:acetic acid (pH 2.8) was efficient at eluting template BHb (with 80 ± 10% removal). Selectivity studies of NHMA BHb-MIPs revealed higher adsorption and selective recognition properties to BHb (64.5 kDa) when compared to non-cognate BSA (66 kDa), myoglobin (Mb, 17.5 kDa), lysozyme (Lyz, 14.7 kDa) thaumatin (Thau, 22 kDa) and trypsin (Tryp, 22.3 kDa). The QCM gave frequency shifts of ∼1500 ± 50 Hz for template BHb rebinding in both AA and NHMA MIPs, whereas AA-based MIPs exhibited an interference signal of ∼2200 ± 50 Hz for non-cognate BSA in comparison to a ∼500 ± 50 Hz shift with NHMA MIPs. Our results show that NHMA-based hydrogel MIP are superior to AA and NIPAM

Automating the application of smart materials for protein crystallization

The fabrication and validation of the first semi-liquid nonprotein nucleating agent to be administered automatically to crystallization trials is reported. This research builds upon prior demonstration of the suitability of molecularly imprinted polymers (MIPs; known as 'smart materials') for inducing protein crystal growth. Modified MIPs of altered texture suitable for high-throughput trials are demonstrated to improve crystal quality and to increase the probability of success when screening for suitable crystallization conditions. The application of these materials is simple, time-efficient and will provide a potent tool for structural biologists embarking on crystallization trials. © 2015, IUCR. All rights reserved