Nonspecific nuclear uptake of anti-MUC1 aptamers by dead cells: the role of cell viability monitoring in aptamer targeting of membrane-bound protein cancer biomarkers.

Most aptamers targeting cell-expressed antigens are intended for in vivo application, however, these sequences are commonly generated in vitro against synthetic oligopeptide epitopes or recombinant proteins. As these in vitro analogues frequently do not mimic the in vivo target within an endogenous environment, the evolved aptamers are often prone to nonspecific binding. The presence of dead cells and cellular debris further complicate aptamer targeting, due to their high nonspecific affinities to single-stranded DNA. Despite these known limitations, assessment of cell viability and/or the removal of dead cells is rarely applied as part of the methodology during in vivo testing of aptamer binding. Furthermore, the extent and route(s) by which dead cells uptake existing aptamers remains to be determined in the literature. For this purpose, the previously reported aptamer sequences 5TR1, 5TR4, 5TRG2 and S22 - enriched against the MUC1 tumour marker of the mucin glycoprotein family - were used as model sequences to evaluate the influence of cell viability and the presence of nontarget cell-expressed protein on aptamer binding to the MUC1 expressing human cancer cell lines MCF-7, Hs578T, SW480, and SW620. From fluorescence microscopy analysis, all tested aptamers demonstrated extensive nonspecific uptake within the nuclei of dead cells with compromised membrane integrities. Using fluorescent-activated cell sorting (FACS), the inclusion of excess double-stranded DNA as a blocking agent showed no effect on nonspecific aptamer uptake by dead cells. Further nonspecific binding to cell-membrane bound and intracellular protein was evident for each aptamer sequence, as assessed by southwestern blotting and FACS. These factors likely contributed to the ∼120-fold greater binding response of the 5TR1 aptamer to dead MCF-7 cells over equivalent live cell populations. The identification of dead cells and cellular debris using viability stains and the subsequent exclusion of these cells from FACS analysis was identified as an essential requirement for the evaluation of aptamer binding specificity to live cell populations of the cancer cell lines MCF-7, Hs578T and SW480. The research findings stress the importance of dead cell uptake and more comprehensive cell viability screening to validate novel aptamer sequences for diagnostic and therapeutic application.

Gold nanoparticle-streptavidin conjugates for rapid and efficient screening of aptamer function in lateral flow sensors using novel CD4-binding aptamers identified through Crossover-SELEX.

To decrease the burden of laborious and reagent-intensive screening of modified aptamers, their binding function requires assessment in assay formats compatible with the end diagnostic application. Here, we report on the use of an alternative and cost-effective approach: a rapid lateral flow assay (LFA) utilising streptavidin-conjugated gold nanoparticles (AuNP) as reporter molecules to screen novel ssDNA aptamers for their ability to detect CD4. Crossover-SELEX was employed to identify CD4-targeting aptamers from a ssDNA library enriched against a recombinant human CD4 protein (hCD4) conjugated to magnetic-beads and to endogenous CD4 expressed by U937 cells. Counter-selection with IgG-conjugated beads and CD4-negative Ramos RA-1 cells was employed. Following SELEX, four sequences (U4, U14, U20 and U26) were selected for candidate screening. Fluorescence confocal microscopy showed comparable localization of the Cy5-labeled aptamer U26, compared to antibodies binding CD4's cytoplasmic domain. Aptamer-hCD4 binding kinetics were evaluated by a qPCR-based magnetic-bead binding assay to unmodified aptamers. U26 exhibited the highest binding affinity (Kd = 2.93 ± 1.03 nM) to hCD4-conjugated beads. Citrate-stabilized gold nanoparticles (mean particle diameter, 10.59 ± 1.81 nm) were functionalized with streptavidin to allow immobilization of biotin-labeled aptamers. Except for U4, the aptamer-gold nanoparticle conjugates (Apt-AuNP) remained stable under physiological conditions with their size (approx. 15 nm) appropriate for use in the LFAs. Lateral-flow based screening was used to evaluate the suitability of the Apt-AuNPs as CD4-detecting reporter molecules by immobilizing hCD4 and flowing the nanoparticle conjugates across the LFA. Using this approach, two novel sequences were identified as being suitable for the detection of hCD4: visual detection at 9 min was obtained using U20 or U26. After 20 min, equivalent colorimetric hCD4 responses were observed between anti-CD4 monoclonal antibody (ΔI = 94.19 ± 3.71), an existing CD4 aptamer F1-62 (ΔI = 90.31 ± 19.31) and U26 (ΔI = 100.14 ± 14.61) LFA's, each demonstrating high specificity to hCD4 compared to IgG. From the above, Crossover-SELEX allowed for the successful identification of ssDNA aptamers able to detect hCD4. Streptavidin-conjugated AuNPs, when bound to candidate aptamers, show potential application here as screening tools for the rapid evaluation of aptamer performance in low-cost lateral flow diagnostics.

Generation and screening of histamine-specific aptamers for application in a novel impedimetric aptamer-based sensor.

Histamine is an important biomarker in both biomedical and food quality assurance sectors. Current methods of monitoring this compound via fluorescent, electrochemical, and enzymatic means have several drawbacks, preventing routine detection. This work reports on the isolation of single-stranded DNA-based, histamine-targeting aptamers generated by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and the characterisation of these candidates via bioinformatics analysis. Aptamer binding affinity was determined by magnetic bead-based enzyme linked oligonucleotide assays, followed by the detection of unmodified histamine at a physiological pH via electrochemical impedance spectroscopy (EIS). Aptamer H47 demonstrated the lowest apparent binding affinity (72.8 ±â€¯13.9 nmol L-1) towards bead immobilised histamine. When immobilised to a gold surface, H47 demonstrated the largest biosensor response (ΔRct = 6.83 ±â€¯2.00) compared to other single-stranded DNA sequences in the presence of dissolved histamine. The H47 EIS aptasensor also displayed a highly selective, concentration-dependent response towards histamine (linear range = 1 µmol L-1 - 5 mmol L-1), compared to other similar small molecules. Possessing an apparent binding affinity, limit of detection and limit of quantification of 7.80 ±â€¯1.70 mmol L-1, 4.83 mmol L-1 and 16.08 mmol L-1, respectively, the H47 EIS aptasensor demonstrates promise towards the development of aptasensors in applications which require the rapid detection of histamine in solution.

Primary partial humeral head resurfacing: outcomes with the HemiCAP implant.

BACKGROUND: Humeral head defects such as degenerative disease or avascular necrosis are often treated with stemmed hemiarthroplasty or total shoulder arthroplasty. Despite its historical and clinical significance, stemmed humeral head replacement poses inherent technical challenges to placing spherical implants at the anatomically correct head height, version, and neck-shaft angle. PURPOSE: The aim of this study was to assess humeral head inlay arthroplasty as a joint-preserving alternative that maintains the individual head-neck-shaft anatomy. Humeral head inlay arthroplasty also allows intraoperative surface mapping and placement of a contoured articular component that is matched to the patient's defect size, location, and individual surface geometry. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: This retrospective case series included 19 patients (20 shoulders), with an average age of 48.9 years (range, 32-58 years; 16 men, 3 women). Preoperative diagnoses were osteoarthritis in 16 shoulders and osteonecrosis in 4 shoulders. Pre- and postoperative evaluations included physical examination, radiographic assessment, the American Shoulder and Elbow Surgeons Standardized Shoulder Assessment Form, the Simple Shoulder Test, a pain visual analog scale, and patient satisfaction rating. RESULTS: The mean follow-up period was 32.7 months (range, 17-66 months). The mean American Shoulder and Elbow Surgeons score improved from 24.1 to 78.8, mean Simple Shoulder Test score from 3.95 to 9.3, mean visual analog scale score from 8.2 to 2.1, mean forward flexion from 100° to 129°, and mean external rotation from 23° to 43° (P < .001 for all). Radiographic follow-up showed no evidence of periprosthetic fracture, component loosening, osteolysis, or device failure. Patient shoulder self-assessment was 90% poor before surgery and improved to 75% good to excellent at last follow-up; 20% of patients self-rated as somewhat good to somewhat poor, and 5% self-rated as poor. Ninety percent of patients were satisfied with the choice of the procedure. Three patients had postoperative complications unrelated to the implants, including a partial rotator cuff tear treated with physical therapy, preexisting glenoid wear treated with arthroscopic debridement and microfracture, and infection complicated by subscapularis rupture requiring several subsequent surgical procedures but with retention of the implant. CONCLUSION: Humeral head inlay arthroplasty is effective in providing pain relief, functional improvement, and patient satisfaction. Rather than delaying shoulder arthroplasty to end-stage osteoarthritis, humeral head inlay arthroplasty is a promising new direction in primary shoulder arthroplasty for younger and active patients with earlier stage disease.

Modified Harrington reconstruction for advanced periacetabular metastatic disease.

BACKGROUND/OBJECTIVES: Destructive metastatic lesions about the acetabulum result in pain and functional limitations. We assessed whether periacetabular reconstruction (PAR) improves quality of life by examining outcome measures of pain, function, and mobility. METHODS: Thirty-seven patients with a mean follow-up of 23.6 months (range, 0.5-112 months) were retrospectively reviewed. All patients underwent modified Harrington reconstruction with periacetabular screws, cement, and total hip arthroplasty. RESULTS: The mean preoperative MSTS score of 14 (47%, 14/30) improved to 20 (67%, 20/30) after the procedure. Thirty-five patients who were alive 1 month after the procedure were reviewed for outcome. Postoperatively, patients reported a significant improvement in pain (P < 0.0001), mobility (P < 0.0385), and function (P < 0.0186). Kaplan-Meier survivorship curves showed 59% implant- and 55% patient survival at 2 years; and 49% implant- and 39% patient survival at 5 years. Complications included infection (16%, 6/37) and instability (16%, 6/37). CONCLUSIONS: Our experience with PAR has shown good results with improved postoperative functional scores. Implants will generally exceed life expectancy and are an option to restore quality of life.