Search

PCR-based techniques are widely used to identify disease causing bacterial and viral pathogens, especially in point-of-care or near-patient clinical settings that require rapid results and sample-to-answer workflows. However, such techniques often fail to differentiate between closely related species that have highly variable genomes. Here, a homogenous (closed-tube) pathogen identification and classification method is described that combines PCR amplification, array-based amplicon sequence verification, and real-time detection using an inverse fluorescence fluorescence-resonance energy transfer technique. The amplification is designed to satisfy the inclusivity criteria and create ssDNA amplicons, bearing a nonradiating quencher moiety at the 5'-terminus, for all the related species. The array includes fluorescent-labeled probes which preferentially capture the variants of the amplicons and classify them through solid-phase thermal denaturing (melt curve) analysis. Systematic primer and probe design algorithms and empirical validation methods are presented and successfully applied to the challenging example of identification of, and differentiation between, closely related human rhinovirus and human enterovirus strains., (© The Author(s) 2018. Published by Oxford University Press.)

Background: Kidney transplantation is the most effective treatment for end-stage renal disease. Sensitization refers to pre-existing antibodies against human leukocyte antigen (HLA) protein and remains a major barrier to successful transplantation. Despite implementation of desensitization strategies, many candidates fail to respond. Our objective was to determine whether measuring B cell repertoires could differentiate candidates that respond to desensitization therapy., Methods: We developed an assay based on high-throughput DNA sequencing of the variable domain of the heavy chain of immunoglobulin genes to measure changes in B cell repertoires in 19 highly HLA-sensitized kidney transplant candidates undergoing desensitization and 7 controls with low to moderate HLA sensitization levels. Responders to desensitization had a decrease of 5% points or greater in cumulated calculated panel reactive antibody (cPRA) levels, and non-responders had no decrease in cPRA., Results: Dominant B cell clones were not observed in highly sensitized candidates, suggesting that the B cells responsible for sensitization are either not present in peripheral blood or present at comparable levels to other circulating B cells. Candidates that responded to desensitization therapy had pre-treatment repertoires composed of a larger fraction of class-switched (IgG and IgA) isotypes compared to non-responding candidates. After B cell depleting therapy, the proportion of switched isotypes increased and the mutation frequencies of the remaining non-switched isotypes (IgM and IgD) increased in both responders and non-responders, perhaps representing a shift in the repertoire towards memory B cells or plasmablasts. Conversely, after transplantation, non-switched isotypes with fewer mutations increased, suggesting a shift in the repertoire towards naïve B cells., Conclusions: Relative abundance of different B cell isotypes is strongly perturbed by desensitization therapy and transplantation, potentially reflecting changes in the relative abundance of memory and naïve B cell compartments. Candidates that responded to therapy experienced similar changes to those that did not respond. Further studies are required to understand differences between these two groups of highly sensitized kidney transplant candidates.

An electroless gold deposition method was used to deposit Au nanotubules within the pores of a polycarbonate template membrane. Membranes containing Au nanotubules with inside diameters of 2 and 3 nm were prepared for these studies. Thiols were chemisorbed to the inside tubule walls in order to change the chemical environment within the tubules. The effect of the chemical environment within the tubules on the transport properties of the tubule-containing membrane was investigated. Membranes modified with HS-C(16)H(33) preferentially transported hydrophobic permeant molecules. When a homologous series of permeant molecules was used, the most hydrophobic permeant was preferentially partitioned into and transported by the HS-C(16)H(33) derivatized membrane. In addition, the effect of alkyl chain length (R), in a homologous series of thiols R-SH, was investigated. Hydrophobic permeant molecules were preferentially partitioned into and transported by membranes containing the largest alkyl group. In contrast, membranes modified with HS-C(2)H(4)OH preferentially transported the more hydrophilic permeant pyridine. Finally, we show here that the HS-C(16)H(33) derivatized membrane can be used to separate hydrophobic species from hydrophilic species.

Recent advances in membrane-based separations include chiral separations, nanofiltration, ion-permselectivity, gas separation and pervaporation. Novel chiral separations using enzymes and chiral surfactants as carrier have been achieved using facilitated-transport membranes. Graft-copolymeric membranes have been constructed that possess pH-controlled ion selectivity, and membranes have been made from gold nanotubules that exhibit electrochemically controlled permselectivity.