The Contamination of Intravenous Fluid by Felt-Tip Marking Pen Ink: A Pilot Study

Objective: The practice of writing directly on infusion bags with felt-tip marking pen was suggested to cause contaminations. Recommendation against such practice has been published by manufacturers and health care authorities. A chromatography-based laboratory experiment was conducted to substantiate the possibility of ink constituents permeation through Polyvinylchloride (PVC) infusion bag. Methods: A Viaflex® intravenous infusion bag was marked with a blue Artline® marking pen ink. Fluid samples were obtained at different time intervals and tested for any contaminations. A gas chromatography with mass spectrometry capability system was used to analyse fluid samples from infusion bag. Results: Five fluid samples were obtained from the infusion bag at 0, 10, 30, 60, 120 minutes after ink exposure. Chromatograms from each sample were compared with a chromatogram from “blank” intravenous solution. There appeared to be no chromatographic evidence of ink constituents present in all intravenous fluid samples. Conclusion: The practice of writing directly on Viaflex® infusion bags with a felt-tip marking pen has not resulted in contamination of intravenous fluid by ink constituents

Secretory factors from OP9 stromal cells delay differentiation and increase the expansion potential of adult erythroid cells in vitro

Abstract Development of in vitro culture systems for the generation of red blood cells is a goal of scientists globally with the aim of producing clinical grade products for transfusion. Although mature reticulocytes can be efficiently generated by such systems, the numbers produced fall short of that required for therapeutics, due to limited proliferative capacity of the erythroblasts. To overcome this hurdle, approaches are required to increase the expansion potential of such culture systems. The OP9 mouse stromal cell line is known to promote haematopoietic differentiation of pluripotent stem cells, however an effect of OP9 cells on erythropoiesis has not been explored. In this study, we show not only OP9 co-culture, but factors secreted by OP9 cells in isolation increase the proliferative potential of adult erythroid cells by delaying differentiation and hence maintaining self-renewing cells for an extended duration. The number of reticulocytes obtained was increased by approximately 3.5-fold, bringing it closer to that required for a therapeutic product. To identify the factors responsible, we analysed the OP9 cell secretome using comparative proteomics, identifying 18 candidate proteins. These data reveal the potential to increase erythroid cell numbers from in vitro culture systems without the need for genetic manipulation or co-culture

Novel Adiponectin Variants Identified in Type 2 Diabetic Patients Reveal Multimerization and Secretion Defects

ADIPOQ, encoding adiponectin, is a candidate gene for type 2 diabetes (T2D) identified by genome-wide linkage analyses with supporting evidence showing the protein function in sensitizing insulin actions. In an endeavor to characterize candidate genes causing T2D in Thai patients, we identified 10 novel ADIPOQ variations, several of which were non-synonymous variations observed only in the patients. To examine the impact of these non-synonymous variations on adiponectin structure and biochemical characteristics, we conducted a structural analysis of the wild-type and variant proteins by in silico modeling and further characterized biochemical properties of the variants with predicted structural abnormalities from the modeling by molecular and biochemical studies. The recombinant plasmids containing wild-type and variant ADIPOQ cDNAs derived from the variations identified by our study (R55H, R112H, and R131H) and previous work (G90S and R112C) were constructed and transiently expressed and co-expressed in cultured HEK293T cells to investigate their oligomerization, interaction, and secretion. We found that the novel R55H variant impaired protein multimerization but it did not exert the effect over the co-expressed wild-type protein while novel R131H variant impaired protein secretion and also affected the co-expressed wild-type protein in a dominant negative fashion. The R131H variant could traffic from the endoplasmic reticulum to the Golgi, trans-Golgi network, and early endosome but could not be secreted. The R131H variant was likely to be degraded through the lysosomal system and inhibition of its degradation rescued the variant protein from secretion defect. We have shown the possibility of using in silico modeling for predicting the effect of amino acid substitution on adiponectin oligomerization. This is also the first report that demonstrates a dominant negative effect of the R131H variant on protein secretion and the possibility of using protein degradation inhibitors as therapeutic agents in the patients carrying adiponectin variants with secretion defect

Smartphone multiplex microcapillary diagnostics using Cygnus: development and evaluation of rapid serotype-specific NS1 detection with dengue patient samples

Laboratory diagnosis of dengue virus (DENV) infection including DENV serotyping requires skilled labor and well-equipped settings. DENV NS1 lateral flow rapid test (LFT) provides simplicity but lacks ability to identify serotype. A simple, economical, point-of-care device for serotyping is still needed. We present a gravity driven, smartphone compatible, microfluidic device using microcapillary film (MCF) to perform multiplex serotype-specific immunoassay detection of dengue virus NS1. A novel device–termed Cygnus–with a stackable design allows analysis of 1 to 12 samples in parallel in 40 minutes. A sandwich enzyme immunoassay was developed to specifically detect NS1 of all four DENV serotypes in one 60-μl plasma sample. This test aims to bridge the gap between rapid LFT and laboratory microplate ELISAs in terms of sensitivity, usability, accessibility and speed. The Cygnus NS1 assay was evaluated with retrospective undiluted plasma samples from 205 DENV infected patients alongside 50 febrile illness negative controls. Against the gold standard RT-PCR, clinical sensitivity for Cygnus was 82% in overall (with 78, 78, 80 and 76% for DENV1-4, respectively), comparable to an in-house serotyping NS1 microplate ELISA (82% vs 83%) but superior to commercial NS1-LFT (82% vs 74%). Specificity of the Cygnus device was 86%, lower than that of NS1-microplate ELISA and NS1-LFT (100% and 98%, respectively). For Cygnus positive samples, identification of DENV serotypes DENV2-4 matched those by RT-PCR by 100%, but for DENV1 capillaries false positives were seen, suggesting an improved DENV1 capture antibody is needed to increase specificity. Overall performance of Cygnus showed substantial agreement to NS1-microplate ELISA (κ = 0.68, 95%CI 0.58–0.77) and NS1-LFT (κ = 0.71, 95%CI 0.63–0.80). Although further refinement for DENV-1 NS1 detection is needed, the advantages of multiplexing and rapid processing time, this Cygnus device could deliver point-of-care NS1 antigen testing including serotyping for timely DENV diagnosis for epidemic surveillance and outbreak prediction

The study of nuclear ribonuclease P in <italic>Saccharomyces cerevisiae</italic> using novel RNA affinity tags.

Ribonuclease P (RNase P) is the enzyme responsible for a cleavage of the 5' leaders of pre-tRNAs in all living organisms. In the yeast Saccharomyces cerevisiae, nuclear RNase P consists of one RNA (RPR1 RNA) and nine protein subunits. RPR1 RNA is initially transcribed as a precursor and subsequently processed into a mature form. Defects in subunit assembly have been associated with a failure of RPR1 RNA to be processed from the precursor to mature form. In order to understand the holoenzyme subunit assembly, the studies in this dissertation address the nature of the precursor form of RNase P (pre-RNase P). Instead of using a long, multistep chromatographic purification of RNase P, which could lead to degradation and loss of the enzymatic activity, a rapid and specific affinity isolation of the enzyme is strongly preferred. However, the RNA affinity ligands for detection and isolation of RNAs or ribonucleoproteins have been lacking. In this study, two small RNA tags have been identified through in vitro selection. These tags bind specifically to Sephadex or streptavidin, and the binding to their targets can be disrupted under mild conditions with either dextran or d-biotin, respectively. This useful feature allows specific isolation and recovery of the tagged RNAs or ribonucleoproteins with intact structures and activity. To isolate RNase P, RPR1 RNA was tagged at various positions with the Sephadex-binding RNA, enabling a rapid and specific affinity isolation of either pre- or mature-RNase P. Pre-RNase P was found to be as active in pre-tRNA cleavage in vitro as mature-RNase P. Its protein subunit composition was also similar to that of mature-RNase P, except that very little Pop3p and Rpr2p could be detected relative to the other subunits. Further depletion of these two subunits did not affect RPR1 RNA maturation even though they are required for RNase P function in vivo. In situ hybridization showed that pre-RNase P RNA was localized primarily in the nucleolus, indicating that the assembly is likely to occur there. The finding that pre-RNase P was enzymatically active suggests that, in addition to being the precursor, it might have an in vivo catalytic role for either pre-tRNAs or other unidentified substrates.Ph.D.BiochemistryBiological SciencesMolecular biologyPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/123287/2/3068971.pd

The Ability of Nuclease-Resistant RNA Aptamer against Streptococcus suis Serotype 2, Strain P1/7 to Reduce Biofilm Formation In Vitro

Streptococcus suis, a Gram-positive bacterium, is an important swine and human pathogen, with serotype 2 being the most prevalent strain found worldwide. Deafness, meningitis, and death (in severe cases) are observed in S. suis-infected cases. Development of the ligands that can bind to S. suis with high affinity and specificity could be beneficial for the diagnosis and treatment of S. suis infection. Herein, the nuclease-resistant RNA aptamers based on 2&prime;-fluoropyrimidine modification against S. suis serotype 2, strain P1/7, were established using the cell- Systematic Evolution of Ligands by Exponential enrichment (SELEX) technique. One of the aptamers, R8-su12, could bind to the S. suis target strain as well as other S. suis serotypes, i.e., 1, 1/2, 9, and 14, but not to other bacteria tested, i.e., S. pneumoniae ATCC 49619, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853. Moreover, the R8-su12 RNA aptamer was also capable of inhibiting the biofilm formation of the S. suis target strain, making it potentially useful for the study of biofilm formation and the treatment of S. suis infection in humans and pigs in the future

Deposition of Triglyceride on Soft Contact Lenses from Lipid-Containing Artificial Tears

Objective: To determine the amount of triglyceride deposition from a lipid-containing artificial tear eye drop (Endura® ) on three different types of contact lenses after one day and one month of simulated use. Methods: Simulated use of artificial tears was performed in vitro on three different types of contact lenses, including: high water content (HW), low water content (LW), and silicone hydrogel (SI) contact lenses. To simulate one day of use, contact lenses were incubated in artificial tear solution for 16 hours. To simulate a one month of use, they underwent repeated cycles of 16-hour incubation in artificial tear solution, lens cleaning, and 8-hour storage in a multipurpose solution daily for 30 days. Triglyceride deposited was extracted and determined. Results: After one day of simulated use, amount of deposited triglyceride was significantly different among 3 types of contact lenses with the highest deposition in SI followed by LW and HW contact lenses; the corresponding values (mean ± SD) were 3.79 ± 0.35, 0.84 ± 0.27, and 0.26 ± 0.17 μg/lens, respectively. Only between SI and HW was it found to be statistically different. After one month of simulated use, deposition on SI lenses was slightly increased (6.56 ± 1.10 μg/lens) with that on LW and HW lenses remaining low (0.10 ± 0.12 and 0.55 ± 0.34 μg/lens, respectively). Conclusion: Triglyceride from lipid-containing artificial tears can absorb into contact lenses, particularly those made of silicone hydrogel, most notably after long periods of use. However, levels of triglyceride deposition are relatively low, when compared to lipid deposition from normal tear films during regular use