Aptamers are single-stranded nucleic acids that bind and recognize targets much like antibodies. Recently, aptamers have garnered increased interest due to their unique properties, including ...inexpensive production, simple chemical modification, and long-term stability. At the same time, aptamers possess similar binding affinity and specificity as their protein counterpart. In this review, we discuss the aptamer discovery process as well as aptamer applications to biosensors and separations. In the discovery section, we describe the major steps of the library selection process for aptamers, called systematic evolution of ligands by exponential enrichment (SELEX). We highlight common approaches and emerging strategies in SELEX, from starting library selection to aptamer-target binding characterization. In the applications section, we first evaluate recently developed aptamer biosensors for SARS-CoV-2 virus detection, including electrochemical aptamer-based sensors and lateral flow assays. Then we discuss aptamer-based separations for partitioning different molecules or cell types, especially for purifying T cell subsets for therapeutic applications. Overall, aptamers are promising biomolecular tools and the aptamer field is primed for expansion in biosensing and cell separation.
In recent decades, peptides, which can possess high potency, excellent selectivity, and low toxicity, have emerged as promising therapeutics for cancer applications. Combined with an improved ...understanding of tumor biology and immuno-oncology, peptides have demonstrated robust antitumor efficacy in preclinical tumor models. However, the translation of peptides with intracellular targets into clinical therapies has been severely hindered by limitations in their intrinsic structure, such as low systemic stability, rapid clearance, and poor membrane permeability, that impede intracellular delivery. In this Review, we summarize recent advances in polymer-mediated intracellular delivery of peptides for cancer therapy, including both therapeutic peptides and peptide antigens. We highlight strategies to engineer polymeric materials to increase peptide delivery efficiency, especially cytosolic delivery, which plays a crucial role in potentiating peptide-based therapies. Finally, we discuss future opportunities for peptides in cancer treatment, with an emphasis on the design of polymer nanocarriers for optimized peptide delivery.
BACKGROUND: The clinical use of frozen plasma (FP) continues to increase, both in prophylactic and in therapeutic settings. In 2004, a systematic review of all published randomized controlled trials ...(RCTs) revealed a lack of evidence that supported the efficacy of FP use. This is an update that includes all new RCTs published since the original review.
STUDY DESIGN AND METHODS: Trials involving transfusion of FP up to July 2011 were identified from searches of MEDLINE, EMBASE, CINAHL, The Cochrane Library, and the UKBTS/SRI Transfusion Evidence Library. Methodologic quality was assessed. The primary outcome measure was the effect of FP on survival.
RESULTS: Twenty‐one new trials were eligible for inclusion. These covered prophylactic and therapeutic FP use in liver disease, in cardiac surgery, for warfarin anticoagulation reversal, for thrombotic thrombocytopenic purpura treatment, for plasmapheresis, and in other settings, including burns, shock, and head injury. The largest number of recent RCTs were conducted in cardiac surgery; meta‐analysis showed no significant difference for FP use for the outcome of 24‐hours postoperative blood loss (weighted mean difference, −35.24 mL; 95% confidence interval, −84.16 to 13.68 mL). Overall, there was no significant benefit for FP use across all the clinical conditions. Only two of the 21 trials fulfilled all the criteria for quality assessment.
CONCLUSION: Combined with the 2004 review, 80 RCTs have investigated FP with no consistent evidence of significant benefit for prophylactic and therapeutic use across a range of indications evaluated. There has been little improvement in the overall methodologic quality of RCTs conducted in the past few years.
Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome ...editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.
The coronavirus disease 2019 (COVID‐19) pandemic has devastated families and disrupted healthcare, economies and societies across the globe. Molecular recognition agents that are specific for ...distinct viral proteins are critical components for rapid diagnostics and targeted therapeutics. In this work, we demonstrate the selection of novel DNA aptamers that bind to the SARS‐CoV‐2 spike glycoprotein with high specificity and affinity (<80 nM). Through binding assays and high resolution cryo‐EM, we demonstrate that SNAP1 (SARS‐CoV‐2 spike protein N‐terminal domain‐binding aptamer 1) binds to the S N‐terminal domain. We applied SNAP1 in lateral flow assays (LFAs) and ELISAs to detect UV‐inactivated SARS‐CoV‐2 at concentrations as low as 5×105 copies mL−1. SNAP1 is therefore a promising molecular tool for SARS‐CoV‐2 diagnostics.
Rapid diagnostics are essential for early detection of COVID‐19 and reduced disease spread. In this study, we discovered the DNA aptamer SNAP1 (SARS‐CoV‐2 spike protein N‐terminal domain binding aptamer 1), which binds with high affinity and specificity to SARS‐CoV‐2 spike protein as well as UV‐inactivated SARS‐CoV‐2 virus.
During the COVID-19 (coronavirus disease 2019) pandemic, several SARS-CoV-2 variants of concern emerged, including the Omicron variant, which has enhanced infectivity and immune invasion. Many ...antibodies and aptamers that bind the spike (S) of previous strains of SARS-CoV-2 either do not bind or bind with low affinity to Omicron S. In this study, we report a high-affinity SARS-CoV-2 Omicron RBD-binding aptamer (SCORe) that binds Omicron BA.1 and BA.2 RBD with nanomolar K D1. We employ aptamers SCORe.50 and SNAP4.74 in a multiplexed lateral flow assay (LFA) to distinguish between Omicron and wild-type S at concentrations as low as 100 pM. Finally, we show that SCORe.50 and its dimerized form SCOReD can neutralize Omicron S-pseudotyped virus infection of ACE2-overexpressing cells by >70%. SCORe therefore has potential applications in COVID-19 rapid diagnostics as well as in viral neutralization.
The COVID-19 pandemic is among the greatest health and socioeconomic crises in recent history. Although COVID-19 vaccines are being distributed, there remains a need for rapid testing to limit viral ...spread from infected individuals. We previously identified the SARS-CoV-2 spike protein N-terminal domain (NTD) binding DNA aptamer 1 (SNAP1) for detection of SARS-CoV-2 virus by aptamer–antibody sandwich enzyme-linked immunoassay (ELISA) and lateral flow assay (LFA). In this work, we identify a new aptamer that also binds at the NTD, named SARS-CoV-2 spike protein NTD-binding DNA aptamer 4 (SNAP4). SNAP4 binds with high affinity (<30 nM) for the SARS-CoV-2 spike protein, a 2-fold improvement over SNAP1. Furthermore, we utilized both SNAP1 and SNAP4 in an aptamer sandwich LFA (AptaFlow), which detected SARS-CoV-2 UV-inactivated virus at concentrations as low as 106 copies/mL. AptaFlow costs <$1 per test to produce, provides results in <1 h, and detects SARS-CoV-2 at concentrations that indicate higher viral loads and a high probability of contagious transmission. AptaFlow is a potential approach for a low-cost, convenient antigen test to aid the control of the COVID-19 pandemic.
The extent and dynamics of animal cell biomass accumulation during mitosis are unknown, primarily because growth has not been quantified with sufficient precision and temporal resolution. Using the ...suspended microchannel resonator and protein synthesis assays, we quantify mass accumulation and translation rates between mitotic stages on a single-cell level. For various animal cell types, growth rates in prophase are commensurate with or higher than interphase growth rates. Growth is only stopped as cells approach metaphase-to-anaphase transition and growth resumes in late cytokinesis. Mitotic arrests stop growth independently of arresting mechanism. For mouse lymphoblast cells, growth in prophase is promoted by CDK1 through increased phosphorylation of 4E-BP1 and cap-dependent protein synthesis. Inhibition of CDK1-driven mitotic translation reduces daughter cell growth. Overall, our measurements counter the traditional dogma that growth during mitosis is negligible and provide insight into antimitotic cancer chemotherapies.
Background Smoking and COPD are risk factors for cardiovascular disease, and the pathogenesis may involve endothelial dysfunction. We tested the hypothesis that endothelium-derived ...epoxyeicosatrienoic acid (EET)-mediated endothelial function is impaired in patients with COPD and that a novel soluble epoxide hydrolase inhibitor, GSK2256294, attenuates EET-mediated endothelial dysfunction in human resistance vessels both in vitro and in vivo. Methods Endogenous and stimulated endothelial release of EETs was assessed in 12 patients with COPD, 11 overweight smokers, and two matched control groups, using forearm plethysmography with intraarterial infusions of fluconazole, bradykinin, and the combination. The effects of GSK2256294 on EET-mediated vasodilation in human resistance arteries were assessed in vitro and in vivo in a phase I clinical trial in healthy overweight smokers. Results Compared with control groups, there was reduced vasodilation with bradykinin ( P = .005), a blunted effect of fluconazole on bradykinin-induced vasodilation ( P = .03), and a trend toward reduced basal EET/dihydroxyepoxyeicosatrienoic acid ratio in patients with COPD ( P = .08). A similar pattern was observed in overweight smokers. In vitro, 10 μM GSK2256294 increased 11,12-EET-mediated vasodilation compared with vehicle (90% ± 4.2% vs 72.6% ± 6.2% maximal dilatation) and shifted the bradykinin half-maximal effective concentration (EC50) (–8.33 ± 0.172 logM vs –8.10 ± 0.118 logM; P = .001 for EC50). In vivo, 18 mg GSK2256294 improved the maximum bradykinin response from 338% ± 46% before a dose to 566% ± 110% after a single dose ( P = .02) and to 503% ± 123% after a chronic dose ( P = .003). Conclusions GSK2256294 attenuates smoking-related EET-mediated endothelial dysfunction, suggesting potential therapeutic benefits in patients with COPD. Trial Registry ClinicalTrials.gov; No.: NCT01762774 ; URL: www.clinicaltrials.gov
The generation of anti-PEG antibodies in response to PEGylated proteins, peptides, and carriers significantly limits their clinical applicability. IgM antibodies mediate the clearance of these ...therapeutics upon repeat injection, resulting in toxicity and hindered therapeutic efficacy. We observed this phenomenon in our polymer platform, virus-inspired polymer for endosomal release (VIPER), which employs pH-sensitive triggered display of a lytic peptide, melittin, to facilitate endosomal escape. While the polymer-peptide conjugate was well tolerated after a single injection, we observed unexpected mortality upon repeat injection. Thus, the goal of this work was to enhance the safety and tolerability of VIPER for frequent dosing. Based on previous reports on anti-PEG antibodies and the adjuvant activity of melittin, we characterized the antibody response to polymer, peptide, and polymer-peptide conjugates after repeat-dosing and measured high IgM titers that bound PEG. By substituting the L-amino acid peptide for its D-amino acid enantiomer, we significantly attenuated the anti-PEG antibody generation and toxicity, permitting repeat-injections. We attempted to rescue mice from L-melittin induced toxicity by prophylactic injection of platelet activating factor (PAF) antagonist CV-6209, but observed minimal effect, suggesting that PAF is not the primary mediator of the observed hypersensitivity response. Overall, we demonstrated that the D-amino acid polymer-peptide conjugates, unlike L-amino acid polymer-peptide conjugates, exhibit good tolerability in vivo, even upon repeat administration, and do not elicit the generation of anti-PEG antibodies.
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•Substituting L-melittin for its enantiomer, D-melittin, enables safe, repeat-dosing of melittin peptide-polymer conjugates•Toxicity against L-melittin micelles in vivo is mediated by anti-PEG IgG and IgM antibodies.•D-melittin micelles do not generate a strong IgG or IgM antibody response in vivo.
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