TM Assay Research Articles

Aptamer-enhanced particle aggregation inhibition assay for simple homogeneous protein detection using DNA aptamer and thermo-responsive magnetic nanoparticles

A simple and sensitive homogeneous protein detection system is required for the early detection of biomarkers. Thermo-responsive magnetic particles (TM) have already been developed to achieve easy bound/free separation at the homogeneous protein detection system, but they are still limited owing to the requirement of secondary antibodies and negatively charged polymers, and it is challenging to control the TM aggregation behavior because of the size of the TM. Therefore, at new method to control TM aggregation behavior that is simple, easy, and highly sensitive is required. In this study, we developed a DNA aptamer-based TM assay as a simple protein detection system without additional secondary molecular recognition elements or negatively charged polymer. In the first attempt, a DNA aptamer was modified on the TM surface, and its aggregation behavior was monitored depending on the target molecule concentration. The TM aggregation rate during the heating process decreased depending on the amount of the DNA aptamer and increased depending on the target protein level. This suggests that the DNA aptamer prevented TM aggregation owing to its negative charge and achieved target protein detection owing to the cancellation of repulsion. Capturable aptamers were used in the TM assay to improve the sensitivity and limit of detection. The designed Capture DNA was modified on the TM surface, and the aptamer was captured in the presence of the target protein through a conformational change. Eventually, Capturable aptamer-based TM assay achieved a sub-nanomolar limit of detection and higher sensitivity than that of our initial investigation. Through this study and the ease of the DNA aptamer design, it was shown that the DNA aptamer-modified TM assay enabled the development of a simple and sensitive homogeneous protein detection system.

Monitoring of Systemic Light-Chain Amyloidosis Patients Using Mass Spectrometry (M-inSight) in Serum

Introduction: Systemic light-chain amyloidosis (AL) is a rare disorder characterized by extracellular deposition of monoclonal light-chain immunoglobulins forming insoluble aggregates in different organs. Kidney, heart and liver are principal organs affected by the disease inducing dysfunctions and failures. In this disease, even a low level of circulating light chain can cause significant damages, making challenging the measure of disease burden via peripheral blood. Although a bone marrow aspiration is required to confirm the diagnosis, the evolution of the disease is usually monitored using free light chains dosage, serum and urine electrophoresis, as well as immunofixation. However, these techniques have demonstrated some limitations in terms of sensitivity. Sebia had recently introduced M-inSight TM, a novel, ultra-sensitive and non-invasive assay for monitoring low level disease on blood using targeted mass spectrometry. In this pilot study, M-inSight TM technology is employed to assess the use of mass spectrometry to monitor AL patients. Methods: 14 AL patients were selected to assess the use of M-InSight to detect and quantify AL light chain. 8/14 patients were selected from the AMYDARA study (NCT02816476), consisting in refractory patients treated with Daratumumab. Serum free light-chain assay (Freelite TM), immunofixation on serum/urine as well as bone marrow collection for DNA (ClonoSeq) and RNA (RACE-RepSeq) immunoglobulin next generation sequencing were used to monitor the disease at baseline and end of treatment (EOT) and compared to M-inSight TM. Additionally, 6/14 multicenter patients with normal free light chain level at diagnosis were selected to evaluate the sensitivity of M-InSight. Total mRNA was extracted from BM samples, retrotranscribed to cDNA and then used for immunoglobulin repertoire high-throughput sequencing assay (RACE-RepSeq). cDNA assembly pipeline using IMGT/HighV-QUEST and Vidjil software were used to construct clonotypes and to identify clonal molecular fingerprints and finally their clonotypic peptides for M-InSight analysis. This technology consists of sequence the AL light chain from serum sample and choose clonotypic peptides from the hypervariable regions which then tracked down in the follow-up sample. Results Clonotypic peptides were identified in all eight patients from the baseline sample, which were then confirmed by mass spectrometry. Out of the 8 AMYDARA patients, 5/8 patients achieved very good partial response (VGPR), 2/8 achieved partial response (PR) and 1/8 resulted in complete response (CR). 6 patients had a decrease of the dFLC ratio measure with Freelite TM of 80% or more. 6 out of these 8 patients had MS data highly concordant with that of the serum free light-chain assay. 2 patients had a strong decrease in dFLC while MS measurements were stable or increased. However, detection of the clonotype using RACE-RepSeqand Clonoseq remained elevated, which was more in line with MS than Freelite TM. All patients with normalization of their dFLC were still detected using M-inSight, even those reaching MRD negativity (1 patient) or low number of cells (~10-5, 2 patients) by ClonoSeq. These results were consistent with the RACE-RepSeq which detected the light chain clonotypes in all patients at EOT. Clonotypic peptides using M-InSight were also detected in all 7 patients with normal free light chain at diagnosis. However, in one bi-clonal patient, peptides from only one of both chains were detected, suggesting that only one clone was secreting light chain. Conclusion The goal of this study was to evaluate the M-InSight technique to monitor the evolution of systemic AL amyloidosis in blood samples. The results showed a high concordance with bone marrow MRD measurement and a higher sensitivity than dFLC to monitor the disease. This study shows that MRD monitoring on blood is feasible, using an ultra-sensitive MS technique, avoiding invasive bone-marrow aspirate. M-inSight can track low level AL disease.

Abstract PD9-05: Prognostic and tamoxifen-predictive effect of PAM50 and ROR score in premenopausal women included in the randomised SBII:2 trial

Abstract PD9-05: Prognostic and tamoxifen-predictive effect of PAM50 and ROR score in premenopausal women included in the randomised SBII:2 trial

Isolation of cancer stem cells from cultured breast cancer cells and xenografted breast tumors based on aldehyde dehydrogenase activity.

The heterogeneity of breast tumors is a major factor in the development, progression, and therapeutic response of breast cancer. In terms of therapy resistance, a subset of tumor cells commonly referred to as cancer stem cells (CSCs) or tumor initiating cells (TICs) have a prominent role. These cells have inherent increased tumorigenicity, self-renewal and differentiation capacity, and mechanisms for chemotherapy and radiation resistance. The importance of CSCs/TICs in cancer makes isolating and studying these cells via reliable methods critical. CSCs/TICs can be enriched for by discrete markers. Increased aldehyde dehydrogenase (ALDH) activity as detected by the AldefluorTM assay is a commonly used method. In this chapter, we describe the detailed methods for identification and isolation of putative CSCs/TICs from cultured cells and xenografted breast tumors using the AldefluorTM assay and describe the importance of the ALDH isoforms in breast cancer.

A new blood‐based biomarker of Aβ clearance: The monocyte Aβ mid‐domain assay

AbstractBackgroundDeficiency in cerebral Amyloid beta (Aβ) clearance is implicated in the pathogenesis of Alzheimer’s disease (AD), and Aβ clearance systems are potential therapeutic targets in AD. Clearance of Aβ is a complex process were phagocytosis and degradation of Aβ are two important components. Innate immune‐linked genetic risk factors contribute to AD, and innate immune cells; microglia in the brain and monocytes/macrophages in the peripheral blood (PB), phagocytose Aβ and contribute to clearance. Our objective was to develop an assay measuring Aβ mid‐domain peptides in relevant biological specimens. The intracellular concentration of peptides containing mid‐domain fragments may reflect phagocytic activity or Aβ degradation efficiency.MethodAn immunoassay on the Quanterix Single Molecule Array (Simoa) platform was developed against an Ab 20‐34 peptide using a proprietary anti‐mid‐domain sheep monoclonal antibody as capture antibody (2A9). The detector reagent consists of an anticomplex antibody using Bio‐Rad HuCAL technology (H3) ensuring the optimal Aβ mid‐domain peptide specificity and assay sensitivity. Monocytes were isolated from 8 ml blood samples using a combination of BD CPT blood sampling tubes and RosetteSep antibodies from StemCell.ResultThe 2A9/H3 immunoassay run on the Simoa platform detected Aβ mid‐domain containing peptides with a lower limit of quantification (LLOQ) of approximately 0,7 pg/ml resulting in measurable levels of the peptides in both CSF and monocyte lysates (figure 1 a).ConclusionAs shown in figure 1 b, the 2A9/H3 combination has a high specificity for mid‐domain restricted peptides. Here we show that as used in a sensitive Simoa‐assay, this assay detects Aβ mid‐domain containing peptides both in CSF and monocyte lysates, and may serve as a tool for PB measurement of Aβ catabolism in both disease progression studies and intervention studies. The PreADx ™ assay is an accurate and highly sensitive immunoassay for quantification of mid domain Aβ peptides in relevant biological fluids and cells. The assay demonstrates high precision with monocyte lysates and linearity in spiking and dilution experiments (not shown).

A novel xenonucleic acid-mediated molecular clamping technology for early colorectal cancer screening.

BackgroundColorectal cancer (CRC) is one of the leading causes of cancer-related death. Early detection is critical to reduce CRC morbidity and mortality. In order to meet this need, we developed a molecular clamping assay called the ColoScape TM assay for early colorectal cancer diagnostics.MethodsNineteen mutations in four genes (APC, KRAS, BRAF and CTNNB1) associated with early events in CRC pathogenesis are targeted in the ColoScapeTM assay. Xenonucleic Acid (XNA)-mediated qPCR clamping technology was applied to minimize the wild-type background amplification in order to improve assay sensitivity of CRC mutation detection. The assay analytical performance was verified and validated, cfDNA and FFPE CRC patient samples were evaluated, and an ROC curve was applied to evaluate its performance.ResultsThe data showed that the assay analytical sensitivity was 0.5% Variant Allele Frequency, corresponding to ~7–8 copies of mutant DNA with 5 ng total DNA input per test. This assay is highly reproducible with intra-assay CV of <3% and inter-assay CV of <5%. We have investigated 380 clinical samples including plasma cfDNA and FFPE samples from patients with precancerous and different stages of CRC. The preliminary assay clinical specificity and sensitivity for CRC cfDNA were: 100% (95% CI, 80.3–97.5%) and 92.2% (95% CI, 94.7–100%), respectively, with AUC of 0.96; 96% specificity (95% CI, 77.6–99.7%) and 92% sensitivity (95% CI, 86.1–95.6%) with AUC of 0.94 for CRC FFPE; 95% specificity (95% CI, 82.5%–99.1%) and 62.5% sensitivity (95% CI, 35.8%–83.7%) with AUC of 0.79 for precancerous lesions cfDNA.ConclusionsThe XNA-mediated molecular clamping assay is a rapid, precise, and sensitive assay for the detection of precancerous lesions cfDNA and CRC cfDNA or FFPE samples.

Structural and Functional Characterization of Phosphoglycosyl Transferases from Human Pathogens

Bacterial virulence and viability are strongly dependent on extracellular glycoconjugates including lipopolysaccharide (LPS), capsular polysaccharide (CPS), wall teichoic acid (WTA) and glycoproteins. While these glycoconjugates are diverse in structure and function, they share a common biosynthetic logic. For example, in Gram‐negative bacteria the biosynthesis of a variety of glycoconjugates are initiated on the cytoplasmic face of the bacterial inner membrane, with a phosphoglycosyl transferase (PGT) catalyzing the first membrane‐committed step of the pathway. PGTs transfer a phosphosugar from a nucleotide‐activated donor substrate onto a polyprenol phosphate such as undecaprenol phosphate (UndP). PGTs comprise two distinct superfamilies – polytopic, with multiple membrane spanning helices, and the more recently described monotopic, which contain an unusual re‐entrant membrane intercalating helix. To date, monotopic PGTs have only been observed in bacteria, and remain underexplored antimicrobial targets. In an effort to understand NDP‐sugar specificity and inform inhibitor design, a library of monotopic PGTs from human pathogens was expressed, purified, and subjected to structural and functional characterization. Both detergent and styrene maleic acid (SMA) copolymers were assessed for solubilization towards the purification and stabilization of these challenging protein targets. These efforts have led to purified samples of diverse monotopic PGTs for activity analyses using the UMP‐Glo ™ assay and crystallization screens, which have afforded reproduceable crystals for further investigation.Support or Funding InformationR01GM131627, F32GM134576

Specific Photocleavage Activity of N‐Methylmesoporphyrin IX on G‐Quadruplex DNA Structures

G‐rich regions, at which G‐quadruplexes (G4) are proposed to form, are associated with promoter regions within proto‐oncogenes and may play a role in cell proliferation and carcinogenesis. The ability to identify the formation of G4 structures in these regions, including the temporal aspects of G‐quadruplex formation, is essential to establish the role of these structures in transcriptional regulation. We have focused on identifying highly selective G4 photocleavage agents as molecular probes for establishing the location and timing of G4 formation. N‐methylmesoporphyrin IX (NMM) has long been known as a G4 ligand; however, there has been little investigation into its DNA photocleavage activity. Here we report the ability of NMM to act as a highly selective photocleavage agent for specific G4 structures. We first studied the interaction of NMM with the G4 structures formed by DNA oligonucleotides containing the c‐MYC promoter or human telomeric (hTel) sequences formed in the presence of either Na+ or K+‐containing buffers using a FRET‐based Tm assay and CD spectroscopy. We employed a fluorescence‐based solution assay for G4 cleavage to establish that NMM effects photocleavage (420 nm) of c‐MYC more efficiently than the hTel G4 under both Na+ and K+ conditions, and effects photocleavage of c‐MYC G4 even under white light irradiation. Using denaturing polyacrylamide gel electrophoresis (PAGE), we confirmed this selective photocleavage and demonstrate that cleavage occurs at specific guanine residues of the c‐MYC G4. In order to determine if the photocleavage proceeds through the generation of reactive oxygen species like singlet oxygen, we carried out photocleavage reactions in the presence of H2O and D2O, and employed a turn‐on fluorescent singlet oxygen sensor to detect 1O2 generation. We find that NMM acts to destabilize specific G4 topologies, but oligonucleotides annealed in the presence of NMM form alternative topologies that are thermally stabilized. NMM photocleavage is highly selective for G4 DNA versus duplex DNA, and the K+‐stabilized c‐MYC G4 is preferentially cleaved compared to the Na+‐stabilized structure and the hTel G4 in either Na+ or K+. PAGE analysis indicates that NMM photocleavage of c‐MYC G4 occurs preferentially at the 3′ G‐tetrad residues. This photocleavage does not appear to be due to 1O2. These results indicate that NMM may be a useful tool for elucidating the role of promoter G‐quadruplex structures.Support or Funding InformationSupport for this work by the Cancer Prevention and Research Institute of Texas (RP160852) and Texas State University is gratefully acknowledged.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparison of Roche and Abbott Cytomegalovirus Quantitative PCR Assays in Allogeneic Hematopoetic Cell Transplant Recipients

BackgroundHuman Cytomegalovirus (CMV) infection is prevalent in patients undergoing transplantion (Tx) and carries significant morbidity and mortality. Quantification of CMV viremia has improved with the use of standardized international units and new commercial real time PCR assays. This study compares the current Roche cobas AmpliPrep/cobas TaqMan CMV test (TM assay) and Abbott Molecular RealTime CMV Investigational Use Only assay (ART assay) for quantification of CMV viremia in patients receiving allogeneic hematopoietic stem cell transplants (aHSCT).MethodsProspective CMV positive patients, planned for aHSCT, were consented prior to Tx and followed weekly up 12 weeks post-transplant (PT) and once ~3 months PT. Matched paired plasma samples were processed and analyzed per manufacturer instructions, Henry Ford clinical laboratory processed the samples using the TM assay and the McKinnon Research Laboratory processed the paired samples using the ART assay. Parametric and non-parametric analyses were conducted as appropriate.ResultsFourteen patients enrolled, 1 patient withdrew after entering, 1 patient died after 9 weeks PT (primary disease). Patients received peripheral blood stem cells and 84.6% received myeloablative chemotherapy. In paired samples, quantifiable CMV by TM and ART assays was detected in 6 (5 treated) vs. 8 of 13 patients respectively. ART assay detected CMV in all patients with positive paired samples, 1 detected in an unpaired sample and in 2 patients missed by TM assay (P = 0.021). Assays also differed in samples with no detection, detectable and quantifiable CMV viremia, with more frequent detection in the ART assay (P = 0.009). Time to quantifiable viremia PT by TM and ART assays was a median of 5 vs. 3 weeks (P = 0.026). Bland-Altman plot shows higher viremia levels quantified using the ART assay (P = 0.023). After week 4 PT, ART assay results inversely correlated with platelets counts (P = 0.013). CMV viremia tended to persist 1.9 weeks longer using ART vs.. TM assay (P = 0.07).ConclusionCMV viremia was quantified earlier, at higher levels and persisted in patients with aHSCT using the ART assay compared with the TM assay. Further study is warranted to determine clinical impact of ART assay on the management of CMV viremia.Disclosures J. Mckinnon, Abbott Molecular: Speaker’s Bureau, Grant recipient and Speaker honorarium D. Lucic, Abbott Molecular: Employee, Salary

Comparison of Roche TaqMan v.2.0, Abbott RealTime and Modified Abbott Residual Viremia Assays in Testing of Routine Samples for Viremia Monitoring in HIV Care

BackgroundRoutine HIV RNA clinical monitoring frequently reports virermia (VL) as detectable, quantifiable <50 or blips >50 c/mL. To assess whether the results represent true increases or just assay variability, we designed comparisons of the Roche COBAS AmpliPrep / COBAS TaqMan HIV-1 Test version 2.0 (TM) and the Abbott RealTime HIV-1 assay (RT) to a residual viremia (RV) assay with a limit of detection of 3 c/mL, the Modified Abbott Residual Viremia assay (MARV).MethodsHIV suppressed patients on stable therapy presenting for routine visits at the HIV clinic in Henry Ford Hospital (HFH) were prospectively enrolled. Blood samples and questionnaires were obtained at each visit. Paired plasma samples were processed and analyzed per manufacturer instructions; HFH clinical laboratory processed the samples using the TM assay and the McKinnon Research Laboratory performed the FDA approved RT assay and RV testing using the MARV assay. Parametric and non-parametric analyses were conducted as indicated.Results124 HIV patients are reported with a mean age of 51, mostly male (84%) and African American (64%). Mean CD4 cell counts were 661 cells/mm3. TM assay results for 196 plasma samples were not detectable (ND) 119 (61%), detectable (DT) 61 (31%) and quantifiable viremia (QV) >20 c/mL 16 (8%). 5 patients had QV over 50 c/mL. 60 tested patients (49.5%) had at least one sample either DT or QV. TM results were not correlated to CD4 cell counts, antiretrovirals, medical conditions or reported adherence (avg. 97%). 174 RT and 187 MARV paired tests were ND on 142 (81%) / 121 (65%), DT on 34 (17%) RT, and quantifiable in 0% / 34 (33%) of the samples respectively. Higher VL detection by the TM was shown compared with the RT (P = 0.005) and MARV (P < 0.001) assays. Mean QV by the MARV was 5.0 vs.. 55 c/mL on the TM assay (P < 0.001). VL for all assays trends correlated when compared for ND, DT and >20 copies/mL (P < 0.001). Bland-Altman plots show higher VL detected by TM as compared with MARV (P < 0.001) and good correlation between RT and MARV assays (P = 0.6).ConclusionHIV viremia is more frequently reported by TM assay as compared with the RT assay and was significantly higher than VL detected by the MARV assay. Over detection of VL by the TM assay may impact clinical decision making and increase cost of care.Disclosures J. Mckinnon, Abbott Molecular: Speaker’s Bureau, Grant recipient, Research grant and Speaker honorarium D. Lucic, Abbott Molecular: Employee, Salary

Effects of inhibition of neural precursor cell-expressed developmentally down regulated 8 gene on biological behavior, and androgen receptor of human prostate cancer cells

Objective To explore the effect of lentivirus-mediated short hairpin RNA (shRNA) silencing neural precursor cell-expressed developmentally down regulated 8 (NEDD8) gene on androgen receptor (AR), prostate specific antigen (PSA), proliferation, apoptosis and invasion of human prostate cancer LNCap and VCaP cells. Methods LNCap and VCaP cells were transfected with lentivirus-mediated NEDD8-shRNA. The mRNA and protein expression of NEDD8, AR and PSA was detected by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting, respectively. The proliferation, apoptosis and invasion abilities were evaluated by cell counting kit-8 (CCK-8) assay, flow cytometry and Transwell TM assay, respectively. Results After transfection with NEDD8-shRNA, NEDD8 mRNA expression in LnCaP and VCaP was (0.11±0.03) fold and (0.16±0.04) fold, respectively, as compared to the blank control group; AR mRNA expression was (0.52±0.04) fold and (0.42±0.05) fold; PSA mRNA expression was (0.49±0.04) fold and (0.59±0.06) fold. NEDD8, AR and PSA protein expression in LnCaP and VCaP was (11.26±2.65)% and (6.26±2.04)%, (30.36±4.35)% and (15.36±3.37)%, and (13.37±2.70)% and (11.36±2.67)%, respectively. As compared with the control groups, the mRNA and protein expression levels of NEDD8, AR and PSA were significantly inhibited after transfection with NEDD8-shRNA (P=0.000). As compared with the control groups, the proliferation of LnCaP and VCaP was significantly inhibited after transfection with NEDD8-shRNA. The apoptosis rate of LNCap and VCaP cells was (15.08±1.23)% and (20.37±1.76)% respectively, which was significantly enhanced as compared with the control groups (P=0.001, P=0.000). After transfection, the number of LnCaP and VCaP cells invading the Matrigel was (55.5±12.5) and (60.8±11.3) respectively, which was significantly reduced as compared with the control groups (P=0.000, P=0.004). Conclusion NEDD8 may downregulate AR at the transcriptional level. The lentivirus-mediated shRNA silencing NEDD8 gene could inhibit the proliferation and invasion, and promote apoptosis of LNCap and VCaP cells. Key words: Prostate cancer; Neural precursor cell-expressed developmentally down regulated factor 8; Androgen receptor; RNA interference

A ruthenium(II)-trithiacyclononane curcuminate complex: Synthesis, characterization, DNA-interaction, and cytotoxic activity

The coordination of ruthenium(II) complexes to anionic oxygen-based donors are very rare. This study describes a simple, one-pot method for obtaining [ruthenium(II)(trithiacyclononane)(curcumin)(S-DMSO)]Cl (1) in 37% yield. The structural characterization of complex 1 by elemental analysis, FT-IR, 1-D and 2-D NMR, ESI+-MS as well as UV–vis and fluorescence spectroscopies are presented. The DNA-melting temperature (Tm) assay shows that salmon sperm DNA (smDNA) in the presence of complex 1 has a higher melting temperature, with ΔTm = 7.4 °C, while in the presence of curcumin the melting temperature remains unaltered. The in vitro cytotoxic activities of curcumin and complex 1 were investigated using the tumor human prostate cell line, PC-3, and the healthy cell line, PNT-2. Complex 1 is innocuous toward normal prostate epithelial cells and, whereas curcumin is toxic, with inhibition rates of ca. 35 and 65% at 50 and 80 μM, respectively. On the tumor cell line PC-3, complex 1 did not cause viability changes, whereas curcumin exhibited dose-dependent inhibition, with ca. 73% inhibition at the highest concentration tested, i.e. 80 μM. This study suggests that coordination with the trithiacyclononane ruthenium(II) scaffold stabilizes the photochemical properties of curcumin and strongly changes its biologic activity.

Abstract 2946: Increased ALDH7A1 expression enhances the resistance to the anticancer drugs and colony formation in lung cancer cell lines

Abstract Background: Aldehyde dehydrogenase (ALDH) forms a superfamily of enzymes that catalyze conversion of aldehydes into carboxylic acids, and they are categorized into 19 families in human. Increased ALDH activity was reported to be a potential marker of cancer stem cell (CSC) in various solid tumors including lung cancer. Furthermore, High ALDH expression has been shown to be involved in drug resistance to conventional cytotoxic drugs. Recent studies revealed that ALDH7A1 is highly expressed in prostate cancer and associated with recurrence in patients with surgically resected non-small-cell lung cancer. However, there is not a fundamental link between ALDH7A1 expression and malignant phenotype, including drug resistance and colony formation. Material and methods: Anticancer drug resistant cell lines were established from lung cancer cell lines (PC-6, DMS53, PC-14, PC-9, NCI-H23 and NCI-H2228 cells) by exposure to various anticancer drug, including 7-ethyl-10-hydroxycamptothesin (SN-38), gemcitabine (GEM), cisplatin (CDDP), etoposide, paclitaxel (TXL), pemetrexed, amrubicin, erlotinib and crizotinib. Protein expression was determined by western blotting and gene expression was examined by RT-PCR. ALDH enzyme activity was measured by ALDEFLOUR TM assay. Proliferation was measured using MTS assay. ALDH7A1encoding plasmid was stably transfected into PC-14 cell. These transfected cells displayed sphere formation on ultralow binding plates and survived for more than 3 weeks. Results: We found that the levels of ALDH7A1 expression were higher in various anticancer drug-resistant lung cancer cell lines compared with the parental cells. ALDH enzyme activity was about 4-9 fold higher in SN-38, TXL, CDDP and GEM resistant cell lines (PC6/SN38, PC6/TXL, PC14/CDDP and PC14/GEM) than the parents cells. Up-regulation of ALDH7A1 expression by using a forced expression vector in PC14 cell altered cytotoxicity to SN-38 and TXL. Furthermore, overexpression of ALDH7A1in PC-14 cells enhanced sphere formation relative to the cells transfected with control vector. Conclusion: These results suggested that increased ALDH7A1 expression in lung cancer cell lines might be one of the mechanisms of acquired resistance to anticancer agents and enhance colony formation. Citation Format: Yuichi Sakamori, Hiroaki Ozasa, Eiji Kunii, Yoshitaka Yagi, Takahiro Tsuji, Takeshi Nomizo, Hiroki Nagai, Young Hak Kim, Ken Maeno, Tetsuya Oguri, Michiaki Mishima. Increased ALDH7A1 expression enhances the resistance to the anticancer drugs and colony formation in lung cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2946.

Evaluating the Metal Tolerance Capacity of Microbial Communities Isolated from Alberta Oil Sands Process Water.

Anthropogenic activities have resulted in the intensified use of water resources. For example, open pit bitumen extraction by Canada’s oil sands operations uses an estimated volume of three barrels of water for every barrel of oil produced. The waste tailings–oil sands process water (OSPW)–are stored in holding ponds, and present an environmental concern as they are comprised of residual hydrocarbons and metals. Following the hypothesis that endogenous OSPW microbial communities have an enhanced tolerance to heavy metals, we tested the capacity of planktonic and biofilm populations from OSPW to withstand metal ion challenges, using Cupriavidus metallidurans, a known metal-resistant organism, for comparison. The toxicity of the metals toward biofilm and planktonic bacterial populations was determined by measuring the minimum biofilm inhibitory concentrations (MBICs) and planktonic minimum inhibitory concentrations (MICs) using the MBEC ™ assay. We observed that the OSPW community and C. metallidurans had similar tolerances to 22 different metals. While thiophillic elements (Te, Ag, Cd, Ni) were found to be most toxic, the OSPW consortia demonstrated higher tolerance to metals reported in tailings ponds (Al, Fe, Mo, Pb). Metal toxicity correlated with a number of physicochemical characteristics of the metals. Parameters reflecting metal-ligand affinities showed fewer and weaker correlations for the community compared to C. metallidurans, suggesting that the OSPW consortia may have developed tolerance mechanisms toward metals present in their environment.

The Effect of Eye Drop Excipients against Acanthamoeba Polyphaga by Alamar Blue TM Assay

The Effect of Eye Drop Excipients against Acanthamoeba Polyphaga by Alamar Blue TM Assay

Comparative Evaluation of Sloppy Molecular Beacon and Dual-Labeled Probe Melting Temperature Assays to Identify Mutations in Mycobacterium tuberculosis Resulting in Rifampin, Fluoroquinolone and Aminoglycoside Resistance.

Several molecular assays to detect resistance to Rifampin, the Fluoroquinolones, and Aminoglycosides in Mycobacterium tuberculosis (M. tuberculosis) have been recently described. A systematic approach for comparing these assays in the laboratory is needed in order to determine the relative advantage of each assay and to decide which ones should be advanced to evaluation. We performed an analytic comparison of a Sloppy Molecular Beacon (SMB) melting temperature (Tm) assay and a Dual labeled probe (DLP) Tm assay. Both assays targeted the M. tuberculosis rpoB, gyrA, rrs genes and the eis promoter region. The sensitivity and specificity to detect mutations, analytic limit of detection (LOD) and the detection of heteroresistance were tested using a panel of 56 clinical DNA samples from drug resistant M. tuberculosis strains. Both SMB and DLP assays detected 29/29 (100%) samples with rpoB RRDR mutations and 3/3 (100%) samples with eis promoter mutations correctly. The SMB assay detected all 17/17 gyrA mutants and 22/22 rrs mutants, while the DLP assay detected 16/17 (94%) gyrA mutants and 12/22 (55%) rrs mutants. Both assays showed comparable LODs for detecting rpoB and eis mutations; however, the SMB assay LODs were at least two logs better for detecting wild type and mutants in gyrA and rrs targets. The SMB assay was also moderately better at detecting heteroresistance. In summary, both assays appeared to be promising methods to detect drug resistance associated mutations in M. tuberculosis; however, the relative advantage of each assay varied under each test condition.

Endogenous anti‐C1q antibodies do not interfere with the C1qScreen™ assay

Dear Editor,For decades, the complement cascade has played a centralrole in antibody identification and compatibility [complement-dependent cytotoxicity (CDC) crossmatches] and for thediagnosis of antibody-mediated rejection (C4d staining onhistology and erythrocyte-bound C4d), and has inspirited newanti-rejection drugs (eculizumab and rC1INH).Interference by complement components is already well-known in the setting of conventional (IgG) Luminex™ assay,where C1r

Heavy/Light Chain Ratio as a Biomarker for Monitoring Patients with IgM Monoclonal Gammopathy and Anti-MAG Neuropathy

Background: Patients with neuropathy associated with anti-myelin-associated glycoprotein (MAG) antibodies are managed in part by the detection and whenever possible the measurement of IgM-component on serum protein electrophoresis (SPE). Hevylite™ is an immunoassay developed to quantify separately IgMκ and IgMλ and to calculate the IgMκ/IgMλ ratio (HLC ratio). We sought to determine whether HLC ratio can be used to assess diagnosis and to evaluate response to therapy in patients with anti-MAG neuropathy compared to the other currently available tests. Methods: In our tertiary care centre, we retrospectively studied a series of patients with anti-MAG neuropathy. Clinical data and serum samples were collected before treatment and between 6 months and one year maximum after treatment onset. Hevylite™ was performed in a second time after including all patients. Results: Forty-four patients were included (30 with monoclonal gammopathy of undetermined significance (MGUS) and 14 with Waldenstrom’s Macroglobulinemia (WM)). Thirty-nine patients (89%) had a monoclonal IgMκ and 5 patients had a monoclonal IgMλ (11%). Before treatment, all patients had abnormal HLC ratio and displayed a monoclonal IgM detected by SPE, with only 24 (55%) having a quantifiable IgM M-spike. Taking into account the 39 patients with monoclonal IgMκ, HLC ratio was significantly higher for patients with WM versus patients with MGUS (n=39; p=0.03). Before/after treatment comparisons revealed no significant variations of M-spike (p=0.13) and a trend to variations of HLC ratios (p=0.06) according to the clinical response. Conclusions: Our study confirms that HLC ratio using Hevylite TM assay is able to distinguish patients with WM versus patients with MGUS. It further provides numerical evaluation in patients with anti-MAG neuropathy, for who serum M-spike is no measurable or/and anti-MAG antibodies are also upper the quantifiable threshold.

Comparison of the Simplexa™ Flu A/B & RSV kit (nucleic acid extraction–dependent assay) and the Prodessa ProFlu+™ assay for detecting influenza and respiratory syncytial viruses

The relative performance of 2 widely used reverse transcription polymerase chain reaction (RT-PCR) assays, the Focus diagnostics Simplexa™ Flu A/B & RSV kit (nucleic acid extraction–dependent assay) and the Prodessa Proflu+™ assay, was evaluated using 735 prospectively and retrospectively collected nasopharyngeal swab specimens. Overall, the assays showed positive and negative agreements of 100% and 99.7% for influenza A, 98.1% and 99.9% for influenza B, and 99.3% and 99.5% for respiratory syncytial virus. The relative analytical sensitivity of the 2 assays was also similar.

P37 Changes in the hydrogen sulfide (H2S) system arising from administration of high fat diet

Atherosclerosis is a progressive disease characterized by endothelial dysfunction and accumulation of lipids, cellular debris and fibrous elements within the intima of the vessel wall. Eventually, atherosclerosis triggers plaque formation, vascular remodeling and luminal obstruction [1] . It is becoming increasingly clear that atherosclerosis is an inflammatory disease, dependent on the interaction between inflammatory cells recruited in response to endothelial lipid accumulation, and to the local, wound-healing response of surrounding vascular smooth muscle cells. H 2 S is a gasomediator synthesized from cysteine predominantly by cystathionine- γ -lyase (CSE) [2] and been reported to exhibit both pro- and anti-inflammatory effects [3] . Administration of high fat diet over an extended period results in the development of a low grade inflammatory response which can predispose to atherosclerosis. In the present work we therefore investigated the role of H 2 S in high fat (HF) fed mice. Mice (C57/Black, male, 25 g) were fed either normally or given a HF diet (16% fat, 12.5% cholesterol and 5% sodium cholic acid) for up to 16 weeks. At the end of 8, 12 or 16 weeks, groups of mice were anaesthetised, blood obtained by cardiac puncture and centrifuged for plasma and heart, liver, kidney and lungs removed for biochemical and histological analyses. H 2 S synthesizing enzyme activity was measured in lung, kidney and liver homogenates incubated with L-cysteine (10 mM) and pyridoxal 5′-phosphate (2 mM) by the zinc trapping spectrophotometric assay. Plasma H 2 S levels were determined by high-performance liquid chromatography (HPLC) [4] . Lung, kidney and liver homogenates were used for western blotting of cystathionine- β -synthetase (CBS), CSE and 3-mercaptopyruvate synthase (3-MST). A range of cytokines and chemokines were also assayed in mouse plasma using a Bio-Plex Pro ™ Assay. Plasma serum amyloid A (SAA) and C-reactive protein (CRP) levels were measured using ELISA kits. Oil red O staining was performed on aorta samples. Compared to normal diet mice, HF mice showed similar H 2 S synthesizing activity for lung (normal mice 0.28 ± 0.04 nmol/mg, n = 7; HF mice 0.22 ± 0.03 nmol/mg, n = 7), and decreased H 2 S synthesizing activity for kidney (normal mice 2.49 ± 0.79 nmol/mg, n = 7; HF mice 1.16 ± 0.32 nmol/mg, n = 7) and liver (normal mice 9.21 ± 2.44 nmol/mg, n = 7; HF mice 1.65 ± 0.34 nmol/mg, n = 7) (p 2 S levels (normal mice 256.7 ± 18.4 nM; HF mice 274.6 ± 17.1 nM, n = 5). For western blots, HF fed mice showed increased CBS in liver and kidney, decreased CSE in liver and lung, decreased 3-MST in liver, but similar expression of 3-MST in kidney and lung. HF fed mice had elevated plasma levels of IL-12 (p40), G-CSF, IL-6 and MCP-1. There was no difference between groups in terms of plasma SAA and CRP. There was little difference in aortic lipid content between groups as assessed histologically by oil red O staining. Taken the data together, we propose that changes in H 2 S metabolism are apparent in these animals prior to the development of frank atherosclerosis. Thus, it is possible that monitoring H 2 S may be useful as a predictive biomarker for atherosclerosis.