The effect of polymer end-group on the formation of styrene – maleic acid lipid particles (SMALPs)

A series of block copolymers comprising styrene and maleic acid (SMA) has been prepared using RAFT polymerisation. RAFT often results in a large hydrophobic alkylthiocarbonylthio end group and this work examines its effect on the solution behaviour of the copolymers. SMA variants with, and without, this end group were synthesised and their behaviour compared with a commercially-available random copolymer of similar molecular weight. Dynamic light scattering and surface tension measurements found the RAFT-copolymers preferentially self-assembled into higher-order aggregates in aqueous solution. Small angle neutron scattering using deuterated styrene varients add support to the accepted model that these agreggates comprise a solvent-protected styrenic core with an acid-rich shell. Replacing the hydrophobic RAFT end group with a more hydrophilic nitrile caused differences in the resulting surface activity, attributed to the ability of the adjoining styrene homoblock to drive aggregation. Each of the copolymers formed SMALP nanodiscs with DMPC lipids, which were found to encapsulate a model membrane protein, gramicidin. However, end group variation affected solubilisition of DPPC, a lipid with a higher phase transition temperature. When using RAFT-copolymers terminated with a hydrophobic group, swelling of the bilayer and greater penetration of the homoblock into the nanodisc core occurred with increasing homoblock length. Conversely, commercial and nitrile-terminated RAFT-copolymers produced nanodisc sizes that stayed constant, instead indicating interaction at the edge of the lipid patch. The results highlight how even minor changes to the copolymer can modify the amphiphilic balance between regions, knowledge useful towards optimising copolymer structure to enhance and control nanodisc formation

Insights into N-calls of mitochondrial DNA sequencing using MitoChip v2.0

Developments in DNA resequencing microarrays include mitochondrial DNA (mtDNA) sequencing and mutation detection. Failure by the microarray to identify a base, compared to the reference sequence, is designated an 'N-call.' This study re-examined the N-call distribution of mtDNA samples sequenced by the Affymetrix MitoChip v.2.0, based on the hypothesis that N-calls may represent insertions or deletions (indels) in mtDNA.We analysed 16 patient mtDNA samples using MitoChip. N-calls by the proprietary GSEQ software were significantly reduced when either of the freeware on-line algorithms ResqMi or sPROFILER was utilized. With sPROFILER, this decrease in N-calls had no effect on the homoplasmic or heteroplasmic mutation levels compared to GSEQ software, but ResqMi produced a significant change in mutation load, as well as producing longer N-cell stretches. For these reasons, further analysis using ResqMi was not attempted. Conventional DNA sequencing of the longer N-calls stretches from sPROFILER revealed 7 insertions and 12 point mutations. Moreover, analysis of single-base N-calls of one mtDNA sample found 3 other point mutations.Our study is the first to analyse N-calls produced from GSEQ software for the MitoChipv2.0. By narrowing the focus to longer stretches of N-calls revealed by sPROFILER, conventional sequencing was able to identify unique insertions and point mutations. Shorter N-calls also harboured point mutations, but the absence of deletions among N-calls suggests that probe confirmation affects binding and thus N-calling. This study supports the contention that the GSEQ is more capable of assigning bases when used in conjunction with sPROFILER

Use of matrix-assisted laser desorption ionization-time of flight mass spectrometry to identify vancomycin-resistant enterococci and investigate the epidemiology of an outbreak

The control of vancomycin-resistant enterococci (VRE) has become an increasing burden on health care resources since their discovery over 20 years ago. Current techniques employed for their detection include time-consuming and laborious phenotypic methods or molecular methods requiring costly equipment and consumables and highly trained staff. An accurate, rapid diagnostic test has the ability to greatly reduce the spread of this organism, which has the ability to colonize patients for long periods, potentially even lifelong. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a technology with the ability to identify organisms in seconds and has shown promise in the identification of other forms of antimicrobial resistance in other organisms. Here we show that MALDI-TOF MS is capable of rapidly and accurately identifying vanB-positive Enterococcus faecium VRE from susceptible isolates. Internal validation of the optimal model generated produced a sensitivity of 92.4% and a specificity of 85.2%. Prospective validation results, following incorporation into the routine laboratory work flow, demonstrated a greater sensitivity and specificity at 96.7% and 98.1%, respectively. In addition, the utilization of MALDI-TOF MS to determine the relatedness of isolates contributing to an outbreak is also demonstrated

Collagen I Modifies Connexin‐43 Hemichannel Activity via Integrin α2β1 Binding in TGFβ1‐Evoked Renal Tubular Epithelial Cells

Chronic Kidney Disease (CKD) is associated with sustained inflammation and progressive fibrosis, changes that have been linked to altered connexin hemichannel‐mediated release of adenosine triphosphate (ATP). Kidney fibrosis develops in response to increased deposition of extracellular matrix (ECM), and up‐regulation of collagen I is an early marker of renal disease. With ECM remodeling known to promote a loss of epithelial stability, in the current study we used a clonal human kidney (HK2) model of proximal tubular epithelial cells to determine if collagen I modulates changes in cell function, via connexin‐43 (Cx43) hemichannel ATP release. HK2 cells were cultured on collagen I and treated with the beta 1 isoform of the pro‐fibrotic cytokine transforming growth factor (TGFβ1) ± the Cx43 mimetic Peptide 5 and/or an anti‐integrin α2β1 neutralizing antibody. Phase microscopy and immunocytochemistry observed changes in cell morphology and cytoskeletal reorganization, whilst immunoblotting and ELISA identified changes in protein expression and secretion. Carboxyfluorescein dye uptake and biosensing measured hemichannel activity and ATP release. A Cytoselect extracellular matrix adhesion assay assessed changes in cell‐substrate interactions. Collagen I and TGFβ1 synergistically evoked increased hemichannel activity and ATP release. This was paralleled by changes to markers of tubular injury, partly mediated by integrin α2β1/integrin‐like kinase signaling. The co‐incubation of the hemichannel blocker Peptide 5, reduced collagen I/TGFβ1 induced alterations and inhibited a positive feedforward loop between Cx43/ATP release/collagen I. This study highlights a role for collagen I in regulating connexin‐mediated hemichannel activity through integrin α2β1 signaling, ahead of establishing Peptide 5 as a potential intervention

Genome-wide association mapping for grain manganese in rice (Oryza sativa L.) using a multi-experiment approach

Funding Information: Acknowledgements This research was partly supported by the US National Science Foundation, Plant Genome Research Program (grant #IOS 0701119 to DES, MLG and SRMP) and The US National Institutes of Health (grant 2P4ES007373 to MLG and DES). PR is a PhD student funded by the Thai Government Scholarship.Peer reviewedPostprin

Genome wide association mapping of grain arsenic, copper, molybdenum and zinc in rice (Oryza sativa L.) grown at four international field sites

Peer reviewedPublisher PD