Study of the Molecular Recognition of Aptamers Selected through Ovarian Cancer Cell-SELEX

Ovarian cancer is the most lethal gynecological malignancy, and the ovarian clear cell carcinoma subtype (OCCA) demonstrates a particularly poor response to standard treatment. Improvements in ovarian cancer outcomes, especially for OCCA, could be expected from a clearer understanding of the molecular pathology that might guide strategies for earlier diagnosis and more effective treatment.Cell-SELEX technology was employed to develop new molecular probes for ovarian cancer cell surface markers. A total of thirteen aptamers with K(d)'s to ovarian cancer cells in the pico- to nanomolar range were obtained. Preliminary investigation of the targets of these aptamers and their binding characteristics was also performed.We have selected a series of aptamers that bind to different types of ovarian cancer, but not cervical cancer. Though binding to other cancer cell lines was observed, these aptamers could lead to identification of biomarkers that are related to cancer

Selection of Aptamers Specific for Adipose Tissue

Obesity has reached epidemic proportions, affecting more than one tenth of the world's population. As such, adipose tissue is being increasingly recognized as an important therapeutic target for obesity and related metabolic disorders. While many potential targets of adipose tissue have been established and drugs developed, very few of those drugs specifically target adipose tissue without affecting other tissue. This results from a limited knowledge of both cell-surface markers and physicochemical traits specific to adipocytes that might otherwise be exploited by circulating drugs.Here we report the use of cell-SELEX technology to select two aptamers that can specifically recognize mature adipocytes: adipo-1 and adipo-8. Adipo-8 shows high affinity for differentiated, mature 3T3-L1 adipocytes with a K(d) value of 17.8±5.1 nM. The binding was sustained upon incubation at 37°C and insulin stimulation, but was lost upon trypsin treatment. The binding ability was also verified on frozen tissue slides with low background fluorescence and isolated adipocytes.Aptamer adipo-8 selected from a random library appears to bind to mature differentiated adipocytes specifically. This aptamer holds great promise as a molecular recognition tool for adipocyte biomarker discovery or for targeted delivery of molecules to adipocytes

Antimicrobial point prevalence surveys in two Ghanaian hospitals : opportunities for antimicrobial stewardship

Background: Improved knowledge regarding antimicrobial use in Ghana is urgently needed to reduce antimicrobial resistance (AMR). This includes point prevalence studies (PPS) in hospitals. Objectives were: (i) provide baseline data in two hospitals (Keta Municipal Hospital – KMH and Ghana Police Hospital – GPH) and identify priorities for improvement; (ii) assess the feasibility of conducting PPS; (iii) compare results with others. Methods: Standard PPS design using the Global PPS paper forms, subsequently transferred to their template. Training undertaken by the Scottish team. Quality indicators included rationale for use; stop and review dates, and guideline compliance. Results: Prevalence of antibiotic use was 65.0% in GPH and 82.0% in KMH. Penicillins and other beta-lactam antibiotics were the most prescribed in both hospitals, with third generation cephalosporins mainly used in GPH. Antibiotic treatment was mainly empirical and commonly administered intravenously (IV), duration was generally short with timely oral switching, and infections were mainly community acquired. Encouragingly, good documentation of the indications for antibiotic use in both hospitals and 50.0-66.0% guideline compliance (although for many indications no guideline existed). In addition, almost all prescribed antibiotics had stop dates and there appeared no missed doses. The duration of use for surgical prophylaxis was generally more than one day (69.0% in GPH and 77.0% in KMH). Conclusions: These two hospitals were the first in Ghana to use the Global PPS system. We found the PPS was feasible and relatively rapid, achieved with limited training. Targets for improvement identified included broad-spectrum antibiotics, duration of treatment and high empiric use

Supporting global antimicrobial stewardship : antibiotic prophylaxis for the prevention of surgical site infection in low and middle income countries (LMICs): a scoping review and meta-analysis

Background: The Scottish Antimicrobial Prescribing Group (SAPG) is supporting two hospitals in Ghana to develop antimicrobial stewardship. Early intelligence gathering suggested that surgical prophylaxis was suboptimal. We reviewed the evidence for use of surgical prophylaxis to prevent surgical site infections (SSI) in low and middle-income countries (LMICs) to inform this work. Methods: MEDLINE, Embase, Cochrane, CINHAL and Google Scholar were searched from inception to 17 February, 2020 for trials, audits, guidelines and systematic reviews in English. Grey literature, websites and reference lists of included studies were searched. Randomised clinical trials reporting incidence of SSI following Caesarian section were included in two meta-analyses. Narrative analysis of studies that explored behaviours and attitudes was conducted. Results: This review included 51 studies related to SSI and timing of antibiotic prophylaxis in LMICs. Incidence of SSIs is higher in LMICs, infection surveillance data is poor and there is a lack of local guidelines for antibiotic prophylaxis. Education to improve appropriate antibiotic prophylaxis is associated with reduction of SSI in LMICs.The random effects pooled mean risk ratio of SSI in Caesarian section was 0.77 (95% CI: 0.51 to 1.17) for pre-incision versus post-incision prophylaxis and 0.89 (95% CI: 0.55 to 1.14) for short versus long duration, Reduction in cost and nurse time was reported in shorter duration SAP. Conclusion: There is scope for improvement but interventions must include local context and address strongly held beliefs. Establishment of local multidisciplinary teams will promote ownership and sustainability of change

DNA Aptamers as Molecular Probes for Colorectal Cancer Study

Understanding the molecular features of specific tumors can increase our knowledge about the mechanism(s) underlying disease development and progression. This is particularly significant for colorectal cancer, which is a heterogeneous complex of diseases developed in a sequential manner through a multistep carcinogenic process. As such, it is likely that tumors with similar characteristics might originate in the same manner and have a similar molecular behavior. Therefore, specific mapping of the molecular features can be potentially useful for both tumor classification and the development of appropriate therapeutic regimens. However, this can only be accomplished by developing high-affinity molecular probes with the ability to recognize specific markers associated with different tumors. Aptamers can most easily meet this challenge based on their target diversity, flexible manipulation and ease of development.Using a method known as cell-based Systematic Evolution of Ligands by Exponential enrichment (cell-SELEX) and colorectal cancer cultured cell lines DLD-1 and HCT 116, we selected a panel of target-specific aptamers. Binding studies by flow cytometry and confocal microscopy showed that these aptamers have high affinity and selectivity. Our data further show that these aptamers neither recognize normal colon cells (cultured and fresh), nor do they recognize most other cancer cell lines tested.The selected aptamers can identify specific biomarkers associated with colorectal cancers. We believe that these probes could be further developed for early disease detection, as well as prognostic markers, of colorectal cancers

Silencing of PTK7 in Colon Cancer Cells: Caspase-10-Dependent Apoptosis via Mitochondrial Pathway

Protein tyrosine kinase-7 (PTK7) is a catalytically inactive receptor tyrosine kinase (RTK). PTK7 is upregulated in many common human cancers, including colon cancer, lung cancer, gastric cancer and acute myeloid leukemia. The reason for this up-regulation is not yet known. To explore the functional role of PTK7, the expression of PTK7 in HCT 116 cells was examined using small interference (siRNA)-mediated gene silencing. Following transfection, the siRNA successfully suppressed PTK7 mRNA and protein expression. Knocking down of PTK7 in HCT 116 cells inhibited cell proliferation compared to control groups and induced apoptosis. Furthermore, this apoptosis was characterized by decreased mitochondrial membrane potential and activation of caspase-9 and -10. Addition of a caspase-10 inhibitor totally blocked this apoptosis, suggesting that caspase-10 may play a critical role in PTK7-knockdown-induced apoptosis, downstream of mitochondria. These observations may indicate a role for PTK7 in cell proliferation and cell apoptosis and may provide a potential therapeutic pathway for the treatment of a variety of cancers

In Vitro Selection of DNA Aptamers to Glioblastoma Multiforme

Aptamer probes for specific recognition of glioblastoma multiforme were generated using a repetitive and broad cell-SELEX-based procedure without negative selection. The 454 sequencing technology was used to monitor SELEX, and bioinformatics tools were used to identify aptamers from high throughput data. A group of aptamers were generated that can bind to target cells specifically with dissociation constants (K-d) in the nanomolar range. Selected aptamers showed high affinity to different types of glioblastoma cell lines while showing little or no affinity to other cancer cell lines. The aptamers generated in this study have potential use in different applications, such as probes for diagnosis and devices for targeted drug delivery, as well as tools for molecular marker discovery for glioblastomas