Semiautomated Electrochemical Melting Curve Analysis Device for the Detection of an Osteoporosis Associated Single Nucleotide Polymorphism in Blood.

The detection of single nucleotide polymorphisms (SNPs) is of increasing importance in many areas including clinical diagnostics, patient stratification for pharmacogenomics, and advanced forensic analysis. In the work reported, we apply a semiautomated system for solid-phase electrochemical melting curve analysis (éMCA) for the identification of the allele present at a specific SNP site associated with an increased risk of bone fracture and predisposition to osteoporosis. Asymmetric isothermal recombinase polymerase amplification using ferrocene labeled forward primers was employed to generate single stranded redox labeled amplicons. In a first approach to demonstrate the proof of concept of combining asymmetric RPA with solid-phase éMCA, a simplified system housing a multielectrode array within a polymeric microsystem, sandwiched between two aluminum plates of a heater device, was used. Sample manipulation through the microfluidic channel was controlled by a syringe pump, and an external Ag/AgCl reference electrode was employed. Individual electrodes of the array were functionalized with four different oligonucleotide probes, each probe equivalent in design with the exception of the middle nucleotide. The isothermally generated amplicons were allowed to hybridize to the surface-tethered probes and subsequently subjected to a controlled temperature ramp, and the melting of the duplex was monitored electrochemically. A clear difference between the fully complementary and a single mismatch was observed. Having demonstrated the proof-of-concept, a device for automated éMCA with increased flexibility to house diverse electrode arrays with internal quasi-gold reference electrodes, higher resolution, and broader melting temperature range was developed and exploited for the detection of SNP hetero/homozygosity. Using the optimized conditions, the system was applied to the identification of the allele present at an osteoporosis associated SNP site, rs2741856, in 10 real fingerprick/venous blood samples, with results validated using Sanger sequencing.

Generic Platform for the Multiplexed Targeted Electrochemical Detection of Osteoporosis-Associated Single Nucleotide Polymorphisms Using Recombinase Polymerase Solid-Phase Primer Elongation and Ferrocene-Modified Nucleoside Triphosphates.

Osteoporosis is a multifactorial disease influenced by genetic and environmental factors, which contributes to an increased risk of bone fracture, but early diagnosis of this disease cannot be achieved using current techniques. We describe a generic platform for the targeted electrochemical genotyping of SNPs identified by genome-wide association studies to be associated with a genetic predisposition to osteoporosis. The platform exploits isothermal solid-phase primer elongation with ferrocene-labeled nucleoside triphosphates. Thiolated reverse primers designed for each SNP were immobilized on individual gold electrodes of an array. These primers are designed to hybridize to the SNP site at their 3'OH terminal, and primer elongation occurs only where there is 100% complementarity, facilitating the identification and heterozygosity of each SNP under interrogation. The platform was applied to real blood samples, which were thermally lysed and directly used without the need for DNA extraction or purification. The results were validated using Taqman SNP genotyping assays and Sanger sequencing. The assay is complete in just 15 min with a total cost of 0.3€ per electrode. The platform is completely generic and has immense potential for deployment at the point of need in an automated device for targeted SNP genotyping with the only required end-user intervention being sample addition.

Ultrasensitive determination of ß-conglutin food allergen by means an aptamer assay based on inductively coupled plasma mass spectrometry detection.

The overall objective of this work is the evaluation of different competitive aptamer assays based on inductively coupled plasma mass spectrometry (ICP-MS) detection for the determination of ß-conglutin (food protein allergen from lupin) in flour samples. To this end, two competitive aptamer assay schemes were developed using either thiolated aptamers chemisorbed onto gold nanoparticles (AuNPs) or biotinylated aptamers linked to streptavidin-AuNPs. The influence of ICP-MS detection mode (i.e., conventional vs single particle) on assay performance was explored. In the case of the thiolated aptamer, the limit of detection (LoD) obtained using the single particle mode was improved 2-fold as compared to the LoD provided by the conventional mode. With regards to the biotinylated aptamer, the use of the conventional mode provided a 5-fold improvement of LoD as compared to that obtained for the single particle one. Using the optimized conditions, the best LoD of 2 pM was obtained with the biotinylated aptamer operating with conventional ICP-MS detection. When compared to previous reports using the same aptamer in a competitive assay, the developed method significantly improved the LoD by at least an order of magnitude. Different flour samples containing lupin were successfully analyzed according to European Conformity guidelines for the analysis of food contaminants.

People With Diabetes and Caregivers Prefer Rescue Glucagon Treatment With a Wider Storage Temperature Range and a Nasal Administration, When Efficacy is Similar: A Discrete Choice Experiment in Spain.

BACKGROUND: Conventional injectable glucagon (IG) and nasal glucagon (NG), both having similar efficacy, are two options for the emergency treatment of severe hypoglycemia in Spain. This study elicited the effect of changes in key attributes on preferences for NG and IG medication profiles of people with diabetes and caregivers in Spain. METHODS: The relative attribute importance (RAI) that participants placed on glucagon preparation, preparation and administration time, delivery method, recovery time, device size, storage temperature, and headache risk was estimated from an online discrete choice experiment. In addition, patients and caregivers were presented with NG and IG profiles that included rates of successful administration; the proportion of participants choosing each profile was summarized. RESULTS: The analysis included 276 adults with diabetes (65% type 1) and 270 caregivers (49% type 1). Overall mean age was 40 years; 51% were female. The most important attributes were storage temperature (RAI [95% confidence interval] = 27.3% [22.9-32.2]) and delivery method (17.4% [13.1-21.9]). Headache risk (16.2% [11.8-20.7]), time to prepare and administer (14.5% [10.1-18.8]), glucagon preparation (11.4% [6.8-15.8]), recovery time (8.9% [4.3-13.3]), and device size (4.3% [0.3-8.8]) were also relevant. When comparing medication profiles, significantly more participants (78%) preferred NG over IG profiles (P < .001). CONCLUSION: Adults with diabetes and caregivers prefer a glucagon treatment with a higher rate of successful administration, wider storage temperature, and nasal delivery method, when efficacy is similar. Participants favored NG over conventional IG as a rescue medication for severe hypoglycemia. This information may help decision-making by payers and treatment discussions between health care professionals and patients.

A Label-Free Gold Nanoparticles-Based Optical Aptasensor for the Detection of Retinol Binding Protein 4.

Retinol-binding protein 4 (RBP4) has been implicated in insulin resistance in rodents and humans with obesity and T2DM, making it a potential biomarker for the early diagnosis of T2DM. However, diagnostic tools for low-level detection of RBP4 are still lagging behind. Therefore, there is an urgent need for the development of T2DM diagnostics that are rapid, cost-effective and that can be used at the point-of-care (POC). Recently, nano-enabled biosensors integrating highly selective optical detection techniques and specificity of aptamers have been widely developed for the rapid detection of various targets. This study reports on the development of a rapid gold nanoparticles (AuNPs)-based aptasensor for the detection of RBP4. The retinol-binding protein aptamer (RBP-A) is adsorbed on the surface of the AuNPs through van der Waals and hydrophobic interactions, stabilizing the AuNPs against sodium chloride (NaCl)-induced aggregation. Upon the addition of RBP4, the RBP-A binds to RBP4 and detaches from the surface of the AuNPs, leaving the AuNPs unprotected. Addition of NaCl causes aggregation of AuNPs, leading to a visible colour change of the AuNPs solution from ruby red to purple/blue. The test result was available within 5 min and the assay had a limit of detection of 90.76 ± 2.81 nM. This study demonstrates the successful development of a simple yet effective, specific, and colorimetric rapid assay for RBP4 detection.

Exploiting the Nucleic Acid Nature of Aptamers for Signal Amplification.

Aptamer-based assays and sensors are garnering increasing interest as alternatives to antibodies, particularly due to their increased flexibility for implementation in alternative assay formats, as they can be employed in assays designed for nucleic acids, such as molecular aptamer beacons or aptamer detection combined with amplification. In this work, we took advantage of the inherent nucleic acid nature of aptamers to enhance sensitivity in a rapid and facile assay format. An aptamer selected against the anaphylactic allergen ß-conglutin was used to demonstrate the proof of concept. The aptamer was generated by using biotinylated dUTPs, and the affinity of the modified aptamer as compared to the unmodified aptamer was determined by using surface plasmon resonance to calculate the dissociation constant (KD), and no significant improvement in affinity due to the incorporation of the hydrophobic biotin was observed. The modified aptamer was then applied in a colorimetric competitive enzyme-linked oligonucleotide assay, where ß-conglutin was immobilized on the wells of a microtiter plate, competing with ß-conglutin free in solution for the binding to the aptamer. The limit of detection achieved was 68 pM, demonstrating an improvement in detection limit of three orders of magnitude as compared with the aptamer simply modified with a terminal biotin label. The concept was then exploited by using electrochemical detection and screen-printed electrodes where detection limits of 326 fM and 7.89 fM were obtained with carbon and gold electrodes, respectively. The assay format is generic in nature and can be applied to all aptamers, facilitating an easy and cost-effective means to achieve lower detection limits.

Novel nandrolone aptamer for rapid colorimetric detection of anabolic steroids.

The illicit use of anabolic androgenic steroids (AAS) as performance-enhancing drugs remains a global issue threatening not only the credibility of competitive sports but also public health due to the well-documented adverse effects they elicit. AAS abuse is not restricted only to professional sports, but also extends to recreational athletes and adolescents as well as in livestock production as growth-promoting agents. Testosterone and nandrolone are among the AAS most frequently exploited. Gas chromatography-mass spectrometry is the reference method for AAS detection, but it is strictly laboratory-based and cannot be performed on-site. The great potential of aptamers in bioanalytical applications and specifically for the development of simple analytical tools suitable for on-site analysis has been extensively documented. In this report, we describe the selection and identification of aptamers binding nandrolone, exhibiting affinity dissociation constants in the low nanomolar range. A label-free colorimetric assay based on gold nanoparticles was developed using one of these novel aptamers for the detection of nandrolone and/or its metabolites. The assay could be deployed for the rapid, on-site, facile and cost-effective screening of samples and provide qualitative visual results with a red to purple/blue color change being indicative of a positive result.

Solid-phase recombinase polymerase amplification using ferrocene-labelled dNTPs for electrochemical detection of single nucleotide polymorphisms.

Hypertrophic cardiomyopathies (HCM) are the principal cause of sudden cardiac death in young athletes and it is estimated that 1 in 500 people have HCM. The aim of this work was to develop an electrochemical platform for the detection of HCM-associated SNP in the Myosin Heavy Chain 7 (MYH7) gene, in fingerprick blood samples. The platform exploits isothermal solid-phase primer elongation using recombinase polymerase amplification with either individual or a combination of four ferrocene-labelled nucleoside triphosphates. Four thiolated reverse primers containing a variable base at their 3' end were immobilised on individual gold electrodes of an array. Following hybridisation with target DNA, solid phase recombinase polymerase amplification was carried out and primer elongation incorporating the ferrocene labelled oligonucleotides was only detected at one of the electrodes, thus facilitating identification of the SNP under interrogation. The assay was applied to the direct detection of the SNP in fingerprick blood samples from eight different individuals, with the results obtained corroborating with next generation sequencing. The ability to be able to robustly identify the SNP using a 10 µL fingerprick sample, demonstrates that SNP discrimination is achieved using low femtomolar (ca. 8 × 105 copies DNA) levels of DNA.

Conversations and Reactions Around Severe Hypoglycaemia (CRASH): Spanish results of a global survey of people with type 1 diabetes or insulin-treated type 2 diabetes and caregivers.

INTRODUCTION: Information on experience/management of severe hypoglycaemic events (SHEs) among people with insulin-treated diabetes (PWD) and caregivers (CGs) providing care to PWD was sought. MATERIALS AND METHODS: An online cross-sectional survey was conducted in eight countries. INCLUSION CRITERIA: PWD (aged≥18 years; self-reported type 1 [T1D] or insulin-treated type 2 [T2D] diabetes; experienced ≥1 SHE [hypoglycaemia requiring external assistance] in past 3 years); CGs (layperson aged ≥18 years; caring for PWD meeting all criteria above except age [≥4 years]). This descriptive analysis provides data from Spain. SHE-associated data relate to the most recent SHE. RESULTS: Across all groups (T1D PWD, n=106; T2D PWD, n=88, T1D CG, n=87; T2D CG, n=96), 76-89% reported that the SHE occurred at home; most common cause was eating less than planned (38-53%). Most usual action during the SHE was to intake carbohydrates (67-84%); glucagon use was low (9-36%). Discussion of the SHE with their healthcare provider (HCP) was reported by 70-75% of PWD. During the SHE, 35-69% of PWD/CGs reported feeling scared, unprepared and/or helpless. CONCLUSIONS: Most SHEs occurred outside the healthcare setting; treatment therefore depends greatly on CGs. SHEs have a negative emotional impact on PWD/CGs, underscoring the need for HCPs to discuss SHEs with PWD/CGs, and to provide tools and strategies to prevent and effectively manage SHEs.

Aptamer Sandwich Assay for the Detection of SARS-CoV-2 Spike Protein Antigen.

The novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) emerged at the end of 2019, resulting in the ongoing COVID-19 pandemic. The high transmissibility of the virus and the substantial number of asymptomatic individuals have led to an exponential rise in infections worldwide, urgently requiring global containment strategies. Reverse transcription-polymerase chain reaction is the gold standard for the detection of SARS-CoV-2 infections. Antigen tests, targeting the spike (S) or nucleocapsid (N) viral proteins, are considered as complementary tools. Despite their shortcomings in terms of sensitivity and specificity, antigen tests could be deployed for the detection of potentially contagious individuals with high viral loads. In this work, we sought to develop a sandwich aptamer-based assay for the detection of the S protein of SARS-CoV-2. A detailed study on the binding properties of aptamers to the receptor-binding domain of the S protein in search of aptamer pairs forming a sandwich is presented. Screening of aptamer pairs and optimization of assay conditions led to the development of a laboratory-based sandwich assay able to detect 21 ng/mL (270 pM) of the protein with negligible cross-reactivity with the other known human coronaviruses. The detection of 375 pg of the protein in viral transport medium demonstrates the compatibility of the assay with clinical specimens. Finally, successful detection of the S antigen in nasopharyngeal swab samples collected from suspected patients further establishes the suitability of the assay for screening purposes as a complementary tool to assist in the control of the pandemic.

Electrochemical Detection of Single-Nucleotide Polymorphism Associated with Rifampicin Resistance in Mycobacterium tuberculosis Using Solid-Phase Primer Elongation with Ferrocene-Linked Redox-Labeled Nucleotides.

Here, we report the electrochemical detection of single-point mutations using solid-phase isothermal primer elongation with redox-labeled oligonucleotides. A single-base mutation associated with resistance to rifampicin, an antibiotic commonly used for the treatment of Mycobacterium tuberculosis, was used as a model system to demonstrate a proof-of-concept of the approach. Four 5'-thiolated primers, designed to be complementary with the same fragment of the target sequence and differing only in the last base, addressing the polymorphic site, were self-assembled via chemisorption on individual gold electrodes of an array. Following hybridization with single-stranded DNA, Klenow (exo-) DNA polymerase-mediated primer extension with ferrocene-labeled 2'-deoxyribonucleoside triphosphates (dNFcTPs) was only observed to proceed at the electrode where there was full complementarity between the surface-tethered probe and the target DNA being interrogated. We tested all four ferrocenylethynyl-linked dNTPs and optimized the ratio of labeled/natural nucleotides to achieve maximum sensitivity. Following a 20 min hybridization step, Klenow (exo-) DNA polymerase-mediated primer elongation at 37 °C for 5 min was optimal for the enzymatic incorporation of a ferrocene-labeled nucleotide, achieving unequivocal electrochemical detection of a single-point mutation in 14 samples of genomic DNA extracted from Mycobacterium tuberculosis strains. The approach is rapid, cost-effective, facile, and can be extended to multiplexed electrochemical single-point mutation genotyping.

Solid-Phase Primer Elongation Using Biotinylated dNTPs for the Detection of a Single Nucleotide Polymorphism from a Fingerprick Blood Sample.

Isothermal recombinase polymerase amplification-based solid-phase primer extension is used for the optical detection of a hypertrophic cardiomyopathy associated single nucleotide polymorphism (SNP) in a fingerprick blood sample. The assay exploits four thiolated primers which have the same sequences with the exception of the 3'-terminal base. Target DNA containing the SNP site hybridizes to all four of the immobilized probes, with primer extension only taking place from the primer containing the terminal base that is complementary to the SNP under interrogation. Biotinylated deoxynucleotide triphosphates are used in the primer extension, allowing postextension addition of streptavidin-poly-horseradish peroxidase to bind to the incorporated biotinylated dNTPs. The signal generated following substrate addition can then be measured optically. The percentage of biotinylated dNTPs and the duration of primer extension is optimized and the system applied to the identification of a SNP in a fingerprick blood sample. A methodology of thermal lysis using a 1 in 5 dilution of the fingerprick blood sample prior to application of 95 °C for 30 s is used to extract genomic DNA, which is directly used as a template for solid-phase primer extension on microtiter plates, followed by optical detection. The SNP in the fingerprick sample was identified and its identity corroborated using ion torrent next generation sequencing. Ongoing work is focused on extension to the multiplexed detection of SNPs in fingerprick and other biological samples.

Real-World Use of Insulin Glargine U100 and U300 in Insulin-Naïve Patients with Type 2 Diabetes Mellitus: DosInGlar Study.

INTRODUCTION: In the EDITION clinical trial programme, patients with type 2 diabetes mellitus (T2DM) receiving insulin glargine (IGlar) U300 required 10-15% more insulin than those receiving IGlar U100. This study sought to determine whether this difference was apparent in real-world practice. METHODS: In this observational, retrospective cohort study, electronic medical records in the Big-Pac® database (Real Life Data) relating to adult insulin-naïve patients with T2DM who initiated IGlar U100 or U300 treatment in Spain in 2016-2017 and remained on treatment for 18 months were selected. IGlar U100- and U300-treated patients were matched 1:1 (propensity score matching). The primary analysis compared changes from baseline in mean daily IGlar dose (U and U/kg) at 6 (± 2), 12 (± 2) and 18 (± 2) months between cohorts (paired t tests). Changes in glycated haemoglobin (HbA1c) and weight were analysed descriptively. RESULTS: The IGlar U100 and U300 cohorts included 556 matched pairs (46.9% female) with the following mean (standard deviation) values at baseline, respectively: age 63.6 (12.8) versus 63.7 (11.9) years; years since diagnosis 9.5 (1.4) versus 9.5 (1.3); HbA1c 8.8 (1.3) versus 8.7 (1.5) %; weight 84.6 (16.9) versus 84.7 (17.1) kg. Mean IGlar dose at baseline was 0.19 U/kg/day (both cohorts). Patients receiving IGlar U300 showed a greater increase from baseline in IGlar dose at 6, 12 and 18 months [mean dose (U/kg/day) 5.1%, 10.3% and 12.8% greater, respectively, in IGlar U300-treated patients]. Mean HbA1c was 8.1% in both cohorts at 18 months. Mean (SD) weight at 18 months with IGlar U100 and IGlar300 was 86.8 (17.0) kg and 85.0 (17.1) kg, respectively. CONCLUSION: In real-world practice, insulin dose was significantly higher in IGlar U300-treated than U100-treated patients at 6, 12 and 18 months, with similar reductions in HbA1c. At equal IGlar price/unit in Spain, the increased dose requirements of IGlar U300 would result in higher costs.

A longitudinal study monitoring the quality of life in a national cohort of older adults in Chile before and during the COVID-19 outbreak.

BACKGROUND: Confinement during the COVID-19 pandemic has placed great stress on older adults, which may be affecting their quality of life. Thus, this study aims to describe the changes in mental and physical health, isolation and loneliness, residence and socioeconomic resources in a national cohort of Chilean older adults before and during the COVID-19 outbreak. It also analyzes the changes in depressive symptoms by changes in the other quality of life indicators before and during the COVID-19 outbreak. Possible methodological biases of telephone surveys in older adults living in non-developed countries are also discussed. METHODS: Between June and September 2020, a random subsample of 720 people who had participated in the face-to-face V National Survey on Quality of Life in Older Adults in Chile conducted at the end of 2019 was followed up by telephone. Descriptive bivariate analyses were performed using t-test and non-parametric tests for independent variables, comparing the baseline sample with the current 2020 follow-up sample during the peak of the pandemic outbreak in Latin America. Furthermore, descriptive bivariate analysis through t-test and non-parametric test for paired samples compared the follow-up subsample at baseline with the not-included sample, examining possible biases of the telephone interview compared with the face-to-face interview. RESULTS: In the panel, there was no variation in self-rated health. The health symptoms that worsened were memory, stomach, and mood problems. Depressive symptoms and anxiety increased; similarly, smartphone users, social contacts, intergenerational co-residence and resilience increased. The telephone follow-up sample had a higher educational level and greater smartphone use than those not included in the subsample. CONCLUSIONS: Although some physical and mental health indicators have worsened during the pandemic, older adults mobilized resources that could allow them to maintain their quality of life, such as improved resilience. Thus, these findings can guide future research and the development of efficient strategies to improve these resources among older adults to ensure wellbeing.

Aptamers against the ß-Conglutin Allergen: Insights into the Behavior of the Shortest Multimeric (Intra)Molecular DNA G-Quadruplex.

In previous work, a 93-mer aptamer was selected against the anaphylactic allergen, ß-conglutin and truncated to an 11-mer, improving the affinity by two orders of magnitude, whilst maintaining the specificity. This 11-mer was observed to fold in a G-quadruplex, and preliminary results indicated the existence of a combination of monomeric and higher-order structures. Building on this previous work, in the current study, we aimed to elucidate a deeper understanding of the structural forms of this 11-mer and the effect of the structure on its binding ability. A battery of techniques including polyacrylamide gel electrophoresis, high-performance liquid chromatography in combination with electrospray ionization time-of-flight mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight, thermal binding analysis, circular dichroism and nuclear magnetic resonance were used to probe the structure of both the 11-mer and the 11-mer flanked with TT- at either the 5' or 3' end or at both ends. The TT-tail at the 5' end hinders stacking effects and effectively enforces the 11-mer to maintain a monomeric form. The 11-mer and the TT- derivatives of the 11-mer were also evaluated for their ability to bind its cognate target using microscale thermophoresis and surface plasmon resonance, and biolayer interferometry confirmed the nanomolar affinity of the 11-mer. All the techniques utilized confirmed that the 11-mer was found to exist in a combination of monomeric and higher-order structures, and that independent of the structural form present, nanomolar affinity was observed.

Synthesis of TiO2/Nanozeolite Composites for Highly Efficient Photocatalytic Oxidation of Propene in the Gas Phase.

In this work, we reported the preparation of composites based on titania (TiO2) and Zeolite Socony Mobil-5 (ZSM-5) nanozeolite, following two approaches (i.e., incorporating the presynthesized zeolite in the synthesis medium of TiO2 and incorporating presynthesized TiO2 in the synthesis medium of ZSM-5). The materials synthesized were characterized by X-ray fluorescence (XRF), X-ray diffraction (XRD), nitrogen adsorption, X-ray photoelectron spectroscopy (XPS), ultraviolet-visible spectroscopy (UV-vis), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy-dispersive X-ray (EDX) spectrometry analysis, and their photocatalytic activities were assessed in the oxidation of propene in the gas phase. It was observed that the synthesis methodology affects the final properties of the composite, which ultimately affected their photocatalytic performance in the studied application. It was found that the Nano-ZSM5/TiO2 composite was the most active among the investigated samples, which was attributed to the intimate contact between the two components of the composite, the preserved properties of the photocatalytic active phase in the final material, and the positive contribution of the nanozeolite by increasing the local concentration of propene.

Yersinia pestis detection using biotinylated dNTPs for signal enhancement in lateral flow assays.

Due to the extreme infectivity of Yersinia pestis it poses a serious threat as a potential biowarfare agent, which can be rapidly and facilely disseminated. A cost-effective and specific method for its rapid detection at extremely low levels is required, in order to facilitate a timely intervention for containment. Here, we report an ultrasensitive method exploiting a combination of isothermal nucleic acid amplification with a tailed forward primer and biotinylated dNTPs, which is performed in less than 30 min. The polymerase chain reaction (PCR) and enzyme linked oligonucleotide assay (ELONA) were used to optimise assay parameters for implementation on the LFA, and achieved detection limits of 45 pM and 940 fM using SA-HRP and SA-polyHRP, respectively. Replacing PCR with isothermal amplification, namely recombinase polymerase amplification, similar signals were obtained (314 fM), with just 15 min of amplification. The lateral flow detection of the isothermally amplified and labelled amplicon was then explored and detection limits of 7 fM and 0.63 fg achieved for synthetic and genomic DNA, respectively. The incorporation of biotinylated dNTPs and their exploitation for the ultrasensitive molecular detection of a nucleic acid target has been demonstrated and this generic platform can be exploited for a multitude of diverse real life applications.

One-Pot SELEX: Identification of Specific Aptamers against Diverse Steroid Targets in One Selection.

Aptamers are well-established biorecognition molecules used in a wide variety of applications for the detection of their respective targets. However, individual SELEX processes typically performed for the identification of aptamers for each target can be quite time-consuming, labor-intensive, and costly. An alternative strategy is proposed herein for the simultaneous identification of different aptamers binding distinct but structurally similar targets in one single selection. This one-pot SELEX approach, using the steroids estradiol, progesterone, and testosterone as model targets, was achieved by combining the benefits of counter-SELEX with the power of next-generation sequencing and bioinformatics analysis. The pools from the last stage of the selection were compared in order to discover sequences with preferential abundance in only one of the pools. This led to the identification of aptamer candidates with potential specificity to a single steroid target. Binding studies demonstrated the high affinity of each selected aptamer for its respective target, and low nanomolar range dissociation constants calculated were similar to those previously reported for steroid-binding aptamers selected using traditional SELEX approaches. Finally, the selected aptamers were exploited in microtiter plate assays, achieving nanomolar limits of detection, while the specificity of these aptamers was also demonstrated. Overall, the one-pot SELEX strategy led to the discovery of aptamers for three different steroid targets in one single selection without compromising their affinity or specificity, demonstrating the power of this approach of aptamer discovery for the simultaneous selection of aptamers against multiple targets.

High Affinity Aptamer for the Detection of the Biogenic Amine Histamine.

The importance of histamine in various physiological functions and its involvement in allergenic responses make this small molecule one of the most studied biogenic amines. Even though a variety of chromatography-based methods have been described for its analytical determination, the disadvantages they present in terms of cost, analysis time, and low portability limit their suitability for in situ routine testing. In this work, we sought to identify histamine-binding aptamers that could then be exploited for the development of rapid, facile, and sensitive assays for histamine detection suitable for point-of-need analysis. A classic SELEX process was designed employing magnetic beads for target immobilization and the selection was completed after ten rounds. Following Next Generation Sequencing of the last selection rounds from both positive and counter selection magnetic beads, several sequences were identified and initially screened using an apta-PCR affinity assay (APAA). Structural and functional characterization of the candidates resulted in the identification of the H2 aptamer. The high binding affinity of the H2 aptamer to histamine was validated using four independent assays ( KD of 3-34 nM). Finally, the H2 aptamer was used for the development of a magnetic beads-based competitive assay for the detection of histamine in both buffer and synthetic urine, achieving very low limits of detection of 18 pM and 76 pM, respectively, while no matrix effects were observed. These results highlight the suitability of the strategy followed for identifying small molecule-binding aptamers and the compatibility of the selected H2 aptamer with the analysis of biological samples, thus facilitating the development of point-of-care devices for routine testing. Ongoing work is focused on extending the application of the H2 aptamer to the detection of spoilage in meat, fish, and beverages, as well as evaluating the affinity of truncated forms of the aptamer.

Impact of mind-body interventions in older adults with mild cognitive impairment: a systematic review.

ABSTRACTBackground:Mind-body interventions have been associated with a range of positive outcomes in older adults with mild cognitive impairment (MCI). The aim of the present study was to review the impact of different non-pharmacological programs based on mind-body intervention for older adults with MCI. METHODS: A comprehensive search method as required by the Cochrane Collaboration has been performed through the following databases: Google Scholar, Science Direct, PubMed, PsycINFO, MEDLINE, EMBASE, CINHAL, Cochrane, Ebsco. We included the studies that evaluated the impact of mind-body interventions such as mindfulness or meditation, yoga, Tai Chi and Qigong on cognitive function and everyday functionality of non-hospitalized adults aged 55 years or over with MCI. RESULTS: Nine studies met the inclusion criteria. Results indicated that mind-body interventions improved cognitive function, everyday activities functioning, and mindfulness, as well as resulting in a moderate reduction in fall risk, depression and stress and lower risk of dementia at one year. CONCLUSION: Several mind-body interventions focused broadly on mindfulness, yoga and Tai Chi training have been studied. This review shows that mind-body interventions improved cognitive function and everyday activities functioning, memory, resilience and mindfulness in older adults with MCI. However, the conclusions faced limitations, such as small sample size, heterogeneity of outcome measures, lack of an active control group and absence of long-term follow up. Further high-quality evidence is needed in order to determine whether mind-body interventions are cost-effective for improving cognitive decline in older adults with MCI and for delaying the rapid progression from MCI to Alzheimer or other types of dementia.