Experimental Validation and Prediction of Super-Enhancers: Advances and Challenges.

Super-enhancers (SEs) are cis-regulatory elements of the human genome that have been widely discussed since the discovery and origin of the term. Super-enhancers have been shown to be strongly associated with the expression of genes crucial for cell differentiation, cell stability maintenance, and tumorigenesis. Our goal was to systematize research studies dedicated to the investigation of structure and functions of super-enhancers as well as to define further perspectives of the field in various applications, such as drug development and clinical use. We overviewed the fundamental studies which provided experimental data on various pathologies and their associations with particular super-enhancers. The analysis of mainstream approaches for SE search and prediction allowed us to accumulate existing data and propose directions for further algorithmic improvements of SEs' reliability levels and efficiency. Thus, here we provide the description of the most robust algorithms such as ROSE, imPROSE, and DEEPSEN and suggest their further use for various research and development tasks. The most promising research direction, which is based on topic and number of published studies, are cancer-associated super-enhancers and prospective SE-targeted therapy strategies, most of which are discussed in this review.

A Straightforward Method for the Development of Positively Charged Gold Nanoparticle-Based Vectors for Effective siRNA Delivery.

The therapeutic potential of short interfering RNA (siRNA) to treat many diseases that are incurable with traditional preparations is limited by the extensive metabolism of serum nucleases, low permeability through biological membrane barriers because of a negative charge, and endosomal trapping. Effective delivery vectors are required to overcome these challenges without causing unwanted side effects. Here, we present a relatively simple synthetic protocol to obtain positively charged gold nanoparticles (AuNPs) with narrow size distribution and the surface modified with Tat-related cell-penetrating peptide. The AuNPs were characterized using TEM and the localized surface plasmon resonance technique. The synthesized AuNPs showed low toxicity in experiments in vitro and were able to effectively form complexes with double-stranded siRNA. The obtained delivery vehicles were used for intracellular delivery of siRNA in an ARPE-19 cell line transfected with secreted embryonic alkaline phosphatase (SEAP). The delivered oligonucleotide remained intact and caused a significant knockdown effect on SEAP cell production. The developed material could be useful for delivery of negatively charged macromolecules, such as antisense oligonucleotides and various RNAs, particularly for retinal pigment epithelial cell drug delivery.

Quantitative Rapid Magnetic Immunoassay for Sensitive Toxin Detection in Food: Non-Covalent Functionalization of Nanolabels vs. Covalent Immobilization.

In this study, we present a novel and ultrasensitive magnetic lateral flow immunoassay (LFIA) tailored for the precise detection of zearalenone, a mycotoxin with significant implications for human and animal health. A versatile and straightforward method for creating non-covalent magnetic labels is proposed and comprehensively compared with a covalent immobilization strategy. We employ the magnetic particle quantification (MPQ) technique for precise detection of the labels and characterization of their functionality, including measuring the antibody sorption density on the particle surface. Through kinetic studies using the label-free spectral phase interferometry, the rate and equilibrium constants for the binding of monoclonal antibodies with free (not bound with carrier protein) zearalenone were determined to be kon = 3.42 × 105 M-1s-1, koff = 7.05 × 10-4 s-1, and KD = 2.06 × 10-9 M. The proposed MPQ-LFIA method exhibits detection limits of 2.3 pg/mL and 7.6 pg/mL when employing magnetic labels based on covalent immobilization and non-covalent sorption, with dynamic ranges of 5.5 and 5 orders, correspondingly. We have successfully demonstrated the effective determination of zearalenone in barley flour samples contaminated with Fusarium graminearum. The ease of use and effectiveness of developed test systems further enhances their value as practical tools for addressing mycotoxin contamination challenges.

Kinetic Analysis of Prostate-Specific Antigen Interaction with Monoclonal Antibodies for Development of a Magnetic Immunoassay Based on Nontransparent Fiber Structures.

Prostate cancer is the second most common cancer diagnosed in men worldwide. Measuring the prostate-specific antigen (PSA) is regarded as essential during prostate cancer screening. Early diagnosis of this disease relapse after radical prostatectomy requires extremely sensitive methods. This research presents an approach to development of an ultrasensitive magnetic sandwich immunoassay, which demonstrates the limit of PSA detection in human serum of 19 pg/mL at a dynamic range exceeding 3.5 orders of concentration. Such attractive performance stems, inter alia, from the kinetic analysis of monoclonal antibodies (mAbs) against free PSA to select the mAbs exhibiting best kinetic characteristics and specificity. The analysis is carried out with a label-free multiplex spectral-correlation interferometry compatible with inexpensive single-use glass sensor chips. The high sensitivity of developed PSA immunoassay is due to electronic quantification of magnetic nanolabels functionalized by the selected mAbs and three-dimension porous filters used as an extended solid phase. The assay is promising for PSA monitoring after radical prostatectomy. The proposed versatile approach can be applied for the rational design of highly sensitive tests for detection of other analytes in many fields, including in vitro diagnostics, veterinary, food safety, etc.