Functionalized Aptamers for Detection of Small-Molecule Targets

Aptamers have recently gained considerable attention for small-molecule detection in diverse applications such as drug identification, medical diagnostics, and environmental monitoring. However, the performance of aptamer-based sensors has been greatly limited by the low target affinity and responsiveness of small-molecule binding aptamers. This dissertation describes several novel aptamer engineering and isolation strategies to remedy this problem. Specifically, we first develop a generally applicable strategy to engineer split aptamers containing two binding domains termed cooperative-binding split aptamers (CBSAs). CBSAs exhibit higher target binding affinity and are far more responsive in terms of target-induced split aptamer assembly compared to single-domain parent split aptamers from which they are derived. Using a cocaine-binding CBSA, we achieve specific fluorescence detection of as low as 50 nM cocaine in 10% saliva within 15 minutes. We then develop a general approach for creating rapid and sensitive CBSA-based enzyme-assisted target recycling (EATR)-amplified small-molecule sensors for sensitive target detection. Using this strategy, we develop a fluorescence assay for dehydroisoandrosterone-3-sulfate which achieves 100-fold enhanced target sensitivity relative to a non-EATR-based assay, and a colorimetric assay for visual detection of low-micromolar concentrations of cocaine. To simplify the sensor development process, , we establish a novel and simple SELEX strategy for directly isolating aptamers with intrinsic dye-displacement functionality which transduce target-binding events into a change of dye absorbance. As a demonstration, we isolate an aptamer against the synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV) that can detect MDPV at concentrations as low as 300 nM in a label-free, rapid, and simple dye-displacement assay. To further control the target-binding spectra of aptamers, we employ a novel parallel-and-serial SELEX strategy to isolate an aptamer binding 12 synthetic cathinones with nanomolar affinity but not 11 non-target compounds that are closely related in structure. Using this aptamer, instantaneous visual detection of synthetic cathinones at nanomolar concentrations in biological samples is achieved. In summary, this work demonstrates the great potential of novel aptamer engineering and isolation strategies in generating functional signal-reporting aptamers for sensitive small molecule detection. Importantly, the strategies described here are generalizable and can be used to develop aptamer-based assays for virtually any small-molecule targets

LGR5 Activates Noncanonical Wnt Signaling and Inhibits Aldosterone Production in the Human Adrenal.

CONTEXT: Aldosterone synthesis and cellularity in the human adrenal zona glomerulosa (ZG) is sparse and patchy, presumably due to salt excess. The frequency of somatic mutations causing aldosterone-producing adenomas (APAs) may be a consequence of protection from cell loss by constitutive aldosterone production. OBJECTIVE: The objective of the study was to delineate a process in human ZG, which may regulate both aldosterone production and cell turnover. DESIGN: This study included a comparison of 20 pairs of ZG and zona fasciculata transcriptomes from adrenals adjacent to an APA (n = 13) or a pheochromocytoma (n = 7). INTERVENTIONS: Interventions included an overexpression of the top ZG gene (LGR5) or stimulation by its ligand (R-spondin-3). MAIN OUTCOME MEASURES: A transcriptome profile of ZG and zona fasciculata and aldosterone production, cell kinetic measurements, and Wnt signaling activity of LGR5 transfected or R-spondin-3-stimulated cells were measured. RESULTS: LGR5 was the top gene up-regulated in ZG (25-fold). The gene for its cognate ligand R-spondin-3, RSPO3, was 5-fold up-regulated. In total, 18 genes associated with the Wnt pathway were greater than 2-fold up-regulated. ZG selectivity of LGR5, and its absence in most APAs, were confirmed by quantitative PCR and immunohistochemistry. Both R-spondin-3 stimulation and LGR5 transfection of human adrenal cells suppressed aldosterone production. There was reduced proliferation and increased apoptosis of transfected cells, and the noncanonical activator protein-1/Jun pathway was stimulated more than the canonical Wnt pathway (3-fold vs 1.3-fold). ZG of adrenal sections stained positive for apoptosis markers. CONCLUSION: LGR5 is the most selectively expressed gene in human ZG and reduces aldosterone production and cell number. Such conditions may favor cells whose somatic mutation reverses aldosterone inhibition and cell loss.This work was supported by MJB is an NIHR Senior Investigator NF-SI-0512–10 052; LHS holds a British Heart Foundation PhD studentship FS/11/35/28871; JZ holds a Cambridge Overseas Trust Scholarship; AEDT is funded by the Wellcome Trust Translational Medicine and Therapeutics program 085 686/Z/08/A, and by Singapore A* program; EABA was supported by the Austin Doyle Award (Servier Australia); LHS, JZ and EABA were additionally supported by the NIHR Cambridge Biomedical Research Centre; GM are funded by MRC Programme Grants RDAG/287 and SKAG/001 awarded to Ashok Venkitaraman.This is the author accepted manuscript. The final version is available from the Endocrine Society via http://dx.doi.org/10.1210/jc.2015-173

Efficacy of roxithromycin with gamma globulin in children with mycoplasma pneumonia and its effect on immunity

Purpose: To determine the efficacy of roxithromycin plus gamma globulin in the treatment of children with mycoplasma pneumonia (MPP) and its effect on immune function.Methods: From January 2019 to January 2021, 100 children with MPP assessed for eligibility in Qingdao Women and Children's Hospital, Shandong Province, China, were recruited and randomized (1:1) to receive either gamma globulin (control group) or roxithromycin plus gamma globulin (study group). Levels of tumor necrosis factor (TNF)-α, immunoglobulin (Ig)A, IgM, and IgG were evaluated. Clinical indices, including fever reduction, cough disappearance, duration of hospital stay, etc were also assessed.Results: The study group had a significantly higher clinical efficacy (88 %) than the control group (68 %) (p < 0.05). After treatment, patients in the study group showed lower levels of tumor necrosis factor (TNF)-α than those in the control group (p < 0.05). The eligible patients given roxithromycin plus gamma globulin showed significantly higher levels of immunoglobulin (Ig)A, IgM, and IgG versus those given gamma globulin alone (p < 0.05). Patients in the study group had a shorter time lapse before fever reduction, cough disappearance, lung sign disappearance, and duration of hospital stay than those in the control group (p < 0.05).Conclusion: Roxithromycin plus gamma globulin demonstrate significant benefits in the treatment of children with MPP by mitigating inflammatory response, enhancing immune function, and also significantly alleviating clinical symptoms. Thus, the combination treatment shows good potentials for use in clinical practice

FOXO3a (Forkhead Transcription Factor O Subfamily Member 3a) Links Vascular Smooth Muscle Cell Apoptosis, Matrix Breakdown, Atherosclerosis, and Vascular Remodeling Through a Novel Pathway Involving MMP13 (Matrix Metalloproteinase 13).

OBJECTIVE: Vascular smooth muscle cell (VSMC) apoptosis accelerates atherosclerosis and promotes breakdown of the extracellular matrix, but the mechanistic links between these 2 processes are unknown. The forkhead protein FOXO3a (forkhead transcription factor O subfamily member 3a) is activated in human atherosclerosis and induces a range of proapoptotic and other transcriptional targets. We, therefore, determined the mechanisms and consequences of FOXO3a activation in atherosclerosis and arterial remodeling after injury. APPROACH AND RESULTS: Expression of a conditional FOXO3a allele (FOXO3aA3ER) potently induced VSMC apoptosis, expression and activation of MMP13 (matrix metalloproteinase 13), and downregulation of endogenous TIMPs (tissue inhibitors of MMPs). mmp13 and mmp2 were direct FOXO3a transcriptional targets in VSMCs. Activation of endogenous FOXO3a also induced MMP13, extracellular matrix degradation, and apoptosis, and MMP13-specific inhibitors and fibronectin reduced FOXO3a-mediated apoptosis. FOXO3a activation in mice with VSMC-restricted FOXO3aA3ER induced MMP13 expression and activity and medial VSMC apoptosis. FOXO3a activation in FOXO3aA3ER/ApoE-/- (apolipoprotein E deficient) mice increased atherosclerosis, increased necrotic core and reduced fibrous cap areas, and induced features of medial degeneration. After carotid artery ligation, FOXO3a activation increased VSMC apoptosis, VSMC proliferation, and neointima formation, all of which were reduced by MMP13 inhibition. CONCLUSIONS: FOXO3a activation induces VSMC apoptosis and extracellular matrix breakdown, in part, because of transcriptional activation of MMP13. FOXO3a activation promotes atherosclerosis and medial degeneration and increases neointima after injury that is partly dependent on MMP13. FOXO3a-induced MMP activation represents a direct mechanistic link between VSMC apoptosis and matrix breakdown in vascular disease

A mendelian randomisation study of the causal effect of exercise intensity on the development of type 2 diabetes

ObjectiveThis study examines the causal effects of varying exercise intensities on type 2 diabetes mellitus (T2D) through Mendelian randomization (MR) analysis, using genetic variants as instrumental variables.MethodsA two-sample MR analysis was performed, employing Inverse Variance Weighted (IVW) as the primary method, supported by weighted median, MR-Egger regression, MR-PRESSO, and MR robustness-adjusted contour scores. Data were obtained from the International Exercise Genetics Database (IEGD) and the Global Diabetes Research Consortium (GRC), encompassing over 150,000 individuals for exercise intensity and around 200,000 T2D patients and controls. SNPs linked to exercise intensity were selected based on genome-wide significance (P < 5 × 10^-8) and linkage disequilibrium criteria (distance >10,000 kb, r^2 < 0.001).ResultsThe IVW analysis suggested that high-intensity exercise might reduce T2D risk, but the association was not statistically significant (OR = 0.667, 95% CI = 0.104–4.255, P = 0.667). The wide confidence interval indicates uncertainty in the effect estimate. Low-intensity exercise showed no significant effect on T2D risk (OR ∼ 1.0). Sensitivity analyses, including weighted median and MR-Egger regression, confirmed no significant association between high-intensity exercise and T2D risk. The MR-PRESSO analysis found no significant outliers, and the global test for pleiotropy was non-significant (P = 0.455). Cochran’s Q test for heterogeneity in the IVW analysis was non-significant (Q = 12.45, P = 0.234), indicating consistency among SNP-derived estimates.ConclusionHigh-intensity exercise potentially reduces T2D risk, but the association is not statistically significant. Further research is needed to understand the complex relationship between exercise intensity and T2D

In vitro isolation of class-specific oligonucleotide-based small-molecule receptors

Class-specific bioreceptors are highly desirable for recognizing structurally similar small molecules, but the generation of such affinity elements has proven challenging. We here develop a novel ‘parallel-and-serial’ selection strategy for isolating class-specific oligonucleotide-based receptors (aptamers) in vitro. This strategy first entails parallel selection to selectively enrich cross-reactive binding sequences, followed by serial selection that enriches aptamers binding to a designated target family. As a demonstration, we isolate a class-specific DNA aptamer against a family of designer drugs known as synthetic cathinones. The aptamer binds to 12 diverse synthetic cathinones with nanomolar affinity and does not respond to 11 structurally similar non-target compounds, some of which differ from the cathinone targets by a single atom. This is the first account of an aptamer exhibiting a combination of broad target cross-reactivity, high affinity and remarkable specificity. Leveraging the qualities of this aptamer, instantaneous colorimetric detection of synthetic cathinones at nanomolar concentrations in biological samples is achieved. Our findings significantly expand the binding capabilities of aptamers as class-specific bioreceptors and further demonstrate the power of rationally designed selection strategies for isolating customized aptamers with desired binding profiles. We believe that our aptamer isolation approach can be broadly applied to isolate class-specific aptamers for various small molecule families

Delta/Lambda Chirality: From Enantiomers to Diastereomers in Heterometallic Complexes with Chelating Ligands

The Δ/Λ chirality observed in octahedral molecules with chelating ligands represents the major group of “chiral-at-metal” complexes. Upon shifting from mononuclear to polynuclear systems with multiple (≥2) chiral centers, not only enantiomers but also diastereomers should be considered. We present the first, to the best of our knowledge, diastereomeric pairs Δ,Δ,Δ/Λ,Λ,Λ (1) and Δ,Δ,Λ/Λ,Λ,Δ (2) of the pentanuclear assembly [MnII(ptac)3–Na-CoIII(acac)3–Na-MnII(ptac)3] (ptac = 1,1,1-trifluoro-5,5-dimethyl-2,4-hexanedionate; acac = acetylacetonate). Diastereomers 1 and 2 were isolated in pure form and found to exhibit distinctly different structural characteristics. Importantly, for compounds that are applied as single-source precursors for the quaternary oxide cathode material P2–Na0.67Mn0.67Co0.33O2, the diastereomers revealed different thermal behaviors in terms of volatility and thermal stability. Unambiguous assignment of the Mn and Co positions in both diastereomers has been confirmed by the synchrotron X-ray resonant diffraction technique. Oxidation states of metal ions have been verified by the synchrotron X-ray fluorescence spectroscopy. The diastereomerization between 1 and 2 is not taking place in the solid state (crystal-to-crystal), as well as in the gas phase. The transformation between two diastereomers was observed in the solutions of noncoordinating solvents and was related to the polarities of the solvents and diastereomeric molecules

“Summer storage and winter furnace” – data center waste heat recovery and utilization system for seasonal heat storage

As Chinese society moves towards a more intelligent and connected society, there is an increasing demand for data centers. Under normal circumstances, datacenter IT equipment runs 24 hours a day, which consumes a lot of power and emits a lot of heat. Therefore, when the data center consumes power, it also needs power refrigeration and the ambient temperature to ensure the normal operation of the data center, resulting in huge resource consumption. Combined with the background of carbon neutrality and the current situation of large heating demand and long heating period in Northeast China, this project proposes to use data center waste heat resources as the heat source of building heating, reduce the power consumption required for data center cooling, improve the utilization efficiency and economic efficiency of data center waste heat, and then reduce the carbon emission of data center and building heating