Superenhancer-driven circRNA Myst4 involves in pulmonary artery smooth muscle cell ferroptosis in pulmonary hypertension.

The abnormal expression of circular RNAs (circRNAs) is emerging as a critical cause in regulation of pathological changes of hypoxic pulmonary hypertension (PH), in which ferroptosis is a new pathological change reported recently. However, how circRNAs regulate ferroptosis remains unclear. Here, we proved a significant decrease in circMyst4 expression in hypoxia. In vitro assays revealed that circMyst4 alleviated hypoxic pulmonary artery smooth muscle cell (PASMC) ferroptosis through directly combing with DDX5 in the nucleus to promote GPX4 mRNA processing and inhibiting the formation of the Eef1a1/ACSL4 complex in the cytoplasm. Additionally, superenhancer (SE) was verified to drive the generation of circMyst4. In vivo assays revealed that circMyst4 inhibited the progression of hypoxic PH. Overall, SE-driven circMyst4 may be a new potential therapeutic target for mediating PASMC ferroptosis through promoting DDX5-regulated GPX4 mRNA processing and inhibiting the binding between Eef1a1 and ACSL4.

Sequence Context in DNA i-Motifs Can Nurture Very Stable and Persistent Kinetic Traps.

I-motifs are non-canonical DNA structures with recognized biological significance and a proven utility in material engineering.  Consequently, understanding and control of i-motif properties is essential to sustain progress across both disciplines.  In this work, we systematically investigate how proximity to the most common form of DNA, a double-stranded duplex, influences the thermodynamic and kinetic properties of adjacent i-motifs.  We demonstrate that double-stranded stems in i-motif loops promote kinetic trapping of very stable and persistent partially folded conformations.  Further, we investigate pathways toward rational control over a folding topology makeup.

"Binding" or "Binding and Switching"? A Perspective on Resolving Conformational Changes of Surface-Attached Biomolecular Receptors.

Understanding the structural reconfiguration of a biomolecular receptor remains a topic of particular interest to the biosensing community. This is because conformationally changing receptors are commonly employed in biosensors to harness their capability to bind specifically to their target. Often, such receptors are attached to surfaces so that binding can be transduced into a measurable response. Doing so, however, can impose constraints on the possible configurations they can adopt. Such constraints can ultimately influence, for example, their receptor-target binding models or their affinity, which is essential to provide the desired analytical performances in biosensors. Motivated by the idea of gaining further insights into the impact of surface attachment on conformationally switching receptors attached to surfaces, we explore here the various surface-based techniques capable of monitoring structural changes. We decided to narrow our survey to techniques that have been applied to the investigation of nucleic acids to provide an overview of their key features. We envision that this will bring a broader perspective of the field and the challenges ahead with the hopes of "finding the switch" in surface-attached biomolecular receptors.

Optical scattering methods for the label-free analysis of single biomolecules.

Single-molecule techniques to analyze proteins and other biomolecules involving labels and tethers have allowed for new understanding of the underlying biophysics; however, the impact of perturbation from the labels and tethers has recently been shown to be significant in several cases. New approaches are emerging to measure single proteins through light scattering without the need for labels and ideally without tethers. Here, the approaches of interference scattering, plasmonic scattering, microcavity sensing, nanoaperture optical tweezing, and variants are described and compared. The application of these approaches to sizing, oligomerization, interactions, conformational dynamics, diffusion, and vibrational mode analysis is described. With early commercial successes, these approaches are poised to have an impact in the field of single-molecule biophysics.

Formulation strategies, preparation methods, and devices for pulmonary delivery of biologics.

Biological products, including vaccines, blood components, and recombinant therapeutic proteins, are derived from natural sources such as humans, animals, or microorganisms and are typically produced using advanced biotechnological methods. The success of biologics, particularly monoclonal antibodies, can be attributed to their favorable safety profiles and target specificity. However, their large molecular size presents significant challenges in drug delivery, particularly in overcoming biological barriers. Pulmonary delivery has emerged as a promising route for administering biologics, offering non-invasive delivery with rapid absorption, high systemic bioavailability, and avoidance of first-pass metabolism. This review first details the anatomy and physiological barriers of the respiratory tract and the associated challenges of pulmonary drug delivery (PDD). It further discusses innovations in PDD, the impact of particle size on drug deposition, and the use of secondary particles, such as nanoparticles, to enhance bioavailability and targeting. The review also explains various devices used for PDD, including dry powder inhalers (DPIs) and nebulizers, highlighting their advantages and limitations in delivering biologics. The role of excipients in improving the stability and performance of inhalation products is also addressed. Since dry powders are considered the suitable format for delivering biomolecules, particular emphasis is placed on the excipients used in DPI development. The final section of the article reviews and compares various dry powder manufacturing methods, clarifying their clinical relevance and potential for future applications in the field of inhalable drug formulation.

Harnessing CRISPR/Cas Systems for DNA and RNA Detection: Principles, Techniques, and Challenges.

The emergence of CRISPR/Cas systems has revolutionized the field of molecular diagnostics with their high specificity and sensitivity. This review provides a comprehensive overview of the principles and recent advancements in harnessing CRISPR/Cas systems for detecting DNA and RNA. Beginning with an exploration of the molecular mechanisms of key Cas proteins underpinning CRISPR/Cas systems, the review navigates the detection of both pathogenic and non-pathogenic nucleic acids, emphasizing the pivotal role of CRISPR in identifying diverse genetic materials. The discussion extends to the integration of CRISPR/Cas systems with various signal-readout techniques, including fluorescence, electrochemical, and colorimetric, as well as imaging and biosensing methods, highlighting their advantages and limitations in practical applications. Furthermore, a critical analysis of challenges in the field, such as target amplification, multiplexing, and quantitative detection, underscores areas requiring further refinement. Finally, the review concludes with insights into the future directions of CRISPR-based nucleic acid detection, emphasizing the potential of these systems to continue driving innovation in diagnostics, with broad implications for research, clinical practice, and biotechnology.

Advances in Genotyping Detection of Fragmented Nucleic Acids.

Single nucleotide variant (SNV) detection is pivotal in various fields, including disease diagnosis, viral screening, genetically modified organism (GMO) identification, and genotyping. However, detecting SNVs presents significant challenges due to the fragmentation of nucleic acids caused by cellular apoptosis, molecular shearing, and physical degradation processes such as heating. Fragmented nucleic acids often exhibit variable lengths and inconsistent breakpoints, complicating the accurate detection of SNVs. This article delves into the underlying causes of nucleic acid fragmentation and synthesizes the strengths and limitations of next-generation sequencing technology, high-resolution melting curves, molecular probes, and CRISPR-based approaches for SNV detection in fragmented nucleic acids. By providing a detailed comparative analysis, it seeks to offer valuable insights for researchers working to overcome the challenges of SNV detection in fragmented samples, ultimately advancing the accurate and efficient detection of single nucleotide variants across diverse applications.

Pre-Defined Stem-Loop Structure Library for the Discovery of L-RNA Aptamers that Target RNA G-Quadruplexes.

L-RNA aptamers have been developed to target G-quadruplexes (G4s) and regulate G4-mediated gene expression. However, the aptamer selection process is laborious and challenging, and aptamer identification is subjected to high failure rate. By analyzing the previously reported G4-binding L-RNA aptamers, we found that the stem-loop (SL) structure is favored by G4 binding. Herein, we present a robust and effective G4-SLSELEX-Seq platform specifically for G4 targets by introducing a pre-defined stem-loop structure library during SELEX process. Using G4-SLSELEX-Seq, we rapidly identified an L-RNA aptamer, L-Apt1-12 for EBNA1 RNA G4 (rG4) in just three selection rounds. L-Apt1-12 maintained the stem-loop structure initially introduced, and possessed a unique G-triplex motif that is important for the strong binding affinity and specificity to EBNA1 rG4. Notably, L-Apt1-12 effectively downregulated endogenous EBNA1 protein expression in human cancer cells and showed selective toxicity towards EBV-positive cancer cells, highlighting its potential for targeted therapy against EBV-associated cancers. Furthermore, we demonstrate the robustness and generality of G4-SLSELEX-Seq by selecting L-RNA aptamers for another two G4 targets-APP rG4 and HCV-1a rG4, also obtaining high-affinity aptamers in three selection rounds. These findings demonstrated G4-SLSELEX-Seq can be a robust and efficient platform for the selection of L-RNA aptamers targeting rG4.

Effective removal of host cell-derived nucleic acids bound to hepatitis B core antigen virus-like particles by heparin chromatography.

Virus-like particles (VLPs) show considerable potential for a wide array of therapeutic applications, spanning from vaccines targeting infectious diseases to applications in cancer immunotherapy and drug delivery. In the context of hepatitis B core antigen (HBcAg) VLPs, a promising candidate for gene delivery approaches, the naturally occurring nucleic acid (NA) binding region is commonly utilized for effective binding of various types of therapeutic nucleic acids (NAther). During formation of the HBcAg VLPs, host cell-derived nucleic acids (NAhc) might be associated to the NA binding region, and are thus encapsulated into the VLPs. Following a VLP harvest, the NAhc need to be removed effectively before loading the VLP with NAther. Various techniques reported in literature for this NAhc removal, including enzymatic treatments, alkaline treatment, and lithium chloride precipitation, lack quantitative evidence of sufficient NAhc removal accompanied by a subsequent high VLP protein recovery. In this study, we present a novel heparin chromatography-based process for effective NAhc removal from HBcAg VLPs. Six HBcAg VLP constructs with varying lengths of the NA binding region and diverse NAhc loadings were subjected to evaluation. Process performance was thoroughly examined through NAhc removal and VLP protein recovery analyses. Hereby, reversed phase chromatography combined with UV/Vis spectroscopy, as well as silica spin column-based chromatography coupled with dye-based fluorescence assay were employed. Additionally, alternative process variants, comprising sulfate chromatography and additional nuclease treatments, were investigated. Comparative analyses were conducted with LiCl precipitation and alkaline treatment procedures to ascertain the efficacy of the newly developed chromatography-based methods. Results revealed the superior performance of the heparin chromatography procedure in achieving high NAhc removal and concurrent VLP protein recovery. Furthermore, nuanced relationships between NA binding region length and NAhc removal efficiency were elucidated. Hereby, the construct Cp157 surpassed the other constructs in the heparin process by demonstrating high NAhc removal and VLP protein recovery. Among the other process variants minimal performance variations were observed for the selected constructs Cp157 and Cp183. However, the heparin chromatography-based process consistently outperformed other methods, underscoring its superiority in NAhc removal and VLP protein recovery.

Guideline No. 456: Prenatal Screening for Fetal Chromosomal Anomalies.

OBJECTIVE: To review the available prenatal aneuploidy screening options and to provide updated clinical guidelines for reproductive care providers. TARGET POPULATION: All pregnant persons receiving counselling and providing informed consent for prenatal screening. BENEFITS, HARMS, AND COSTS: Implementation of the recommendations in this guideline should increase clinician competency to offer counselling for prenatal screening options and provide appropriate interventions. Given the variety of available options for prenatal screening with different performance, cost, and availability across Canada, appropriate counselling is of paramount importance to offer the best individual choice to Canadian pregnant persons. Prenatal screening may cause anxiety, and the decisions about prenatal diagnostic procedures are complex given the potential risk of fetal loss. EVIDENCE: Published literature was retrieved through searches of Medline, PubMed, and the Cochrane Library in and prior to July 2023, using an appropriate controlled vocabulary (prenatal diagnosis, amniocentesis, chorionic villi sampling, non-invasive prenatal screening) and key words (prenatal screening, prenatal genetic counselling). Results were restricted to systematic reviews, randomized control trials/controlled clinical trials, and observational studies written in English and published from January 1995 to July 2023. VALIDATION METHODS: The authors rated the quality of evidence and strength of recommendations using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. See Appendix A (Tables A1 for definitions and A2 for interpretations). INTENDED AUDIENCE: Health care providers involved in prenatal screening, including general practitioners, obstetricians, midwives, maternal-fetal medicine specialists, geneticists, and radiologists. SOCIAL MEDIA ABSTRACT: Non-invasive prenatal screening is the most accurate method for detecting major aneuploidies. It is not universally available in the public health system and has some limitations.

A Tetrahedral Framework DNA-Based Bioswitchable miR-150 Delivery System for Sepsis.

Sepsis is a disease with high morbidity and mortality, for which effective treatments are lacking. In recent years, microRNAs (miRs) have been shown to regulate numerous biological processes and can function as therapeutic options for various diseases. However, the poor stability and cell entry properties of miRs have greatly limited their clinical application. In this study, we developed a tetrahedral framework nucleic acid (tFNA)-based bioswitchable miR delivery system (BiRDS) to deliver miR-150 for the treatment of sepsis. BiRDS showed anti-inflammatory effects both in vitro and in vivo by regulating the NF-κB and Notch1 pathways. Therefore, this system holds promise as an ideal candidate for tackling systemic inflammation and multiorgan dysfunction in septic patients in the future.

Nucleic acid liquid biopsies in cardiovascular disease: Cell-free DNA liquid biopsies in cardiovascular disease.

Cardiovascular disease (CVD) is the leading cause of death worldwide, and despite treatment efforts, cardiovascular function cannot always be restored, and progression of disease be prevented. Critical insights are oftentimes based on tissue samples. Current knowledge of tissue pathology typically relies on invasive biopsies or postmortem samples. Liquid biopsies, which assess circulating mediators to deduce the histology and pathology of distant tissues, have been advancing rapidly in cancer research and offer a promising approach to be translated to the understanding and treatment of CVD. The widely understood elevations in cell-free DNA during acute and chronic cardiovascular conditions, associate with disease, severity, and offer prognostic value. The role of neutrophil extracellular traps (NETs) and circulating nucleases in thrombosis provide a solid rationale for liquid biopsies in CVD. cfDNA originates from various tissue types and cellular sources, including mitochondria and nuclei, and can be used to trace cell and tissue type lineage, as well as to gain insight into the activation status of cells. This article discusses the origin, structure, and potential utility of cfDNA, offering a deeper and less invasive approach for the understanding of the complexities of CVD.

Nanomedicines modulate the tumor immune microenvironment for cancer therapy.

INTRODUCTION: In recent years, the evolution of immunotherapy as a means to trigger a robust antitumor immune response has revolutionized cancer treatment. Despite its potential, the effectiveness of cancer immunotherapy is hindered by low response rates and significant systemic side effects. Nanotechnology emerges as a promising frontier in shaping the future of cancer immunotherapy. AREAS COVERED: This review elucidates the pivotal role of nanomedicine in reshaping the immune tumor microenvironment and explores innovative strategies pursued by diverse research groups to enhance the therapeutic efficacy of cancer immunotherapy. It discusses the hurdles encountered in cancer immunotherapy and the application of nanomedicine for small molecule immune modulators and nucleic acid therapeutics. It also highlights the advancements in DNA and mRNA vaccines facilitated by nanotechnology and outlines future trajectories in this evolving field. EXPERT OPINION: Collectively, the integration of nanomedicine into cancer immunotherapy stands as a promising avenue to tackle the intricacies of the immune tumor microenvironment. Innovations such as immune checkpoint inhibitors and cancer vaccines have shown promise. Future developments will likely optimize nanoparticle design through artificial intelligence and create biocompatible, multifunctional nanoparticles, promising more effective, personalized, and durable cancer treatments, potentially transforming the field in the foreseeable future.

Pathologist Interrater Reliability and Clinical Implications of Elevated Donor-Derived Cell-Free DNA beyond Heart Transplant Rejection.

BACKGROUND: There is significant variability amongst pathologists in the histopathological interpretation of the endomyocardial biopsy (EMB) for acute cellular rejection (ACR) and assessment of variability in the interpretation of antibody-mediated rejection (AMR) has not been reported. In contemporary practice, the strategy of allograft surveillance with donor-derived cell-free DNA (dd-cfDNA) as compared to EMB has not been compared with a focus on long-term clinical outcomes beyond acute rejection (AR). METHODS: The Genomic Research Alliance for Transplantation (GRAfT) is a multicenter, prospective cohort study that enrolled patients from 2015 to 2020. The center pathologist read was compared to two blinded core cardiac pathologists. ACR and AMR were graded based on the International Society for Heart and Lung Transplantation (ISHLT) criteria. Weighted Cohen's kappa (κ) was used to evaluate interrater reliability between the center and core reads. To assess long-term outcomes, we evaluated a composite of AR, allograft dysfunction, and mortality within 1 year. RESULTS: The study included 94 patients (median age 55 years [IQR 45, 62]), 30% female, 41% Black race) with a total of 429 EMBs and paired dd-cfDNA measures. The concordance rate between center and core pathologists was 77% for ACR (95%CI: 66% - 89%) and 63% for AMR (95%CI: 53% - 74%). 46 patients had an elevation in dd-cfDNA without AR by EMB. The median dd-cfDNA was 0.49% (IQR: 0.35, 1.01) and subsequent AR, allograft dysfunction, or mortality occurred in 59% of these patients at 1 year. In patients with AR by EMB and negative dd-cfDNA (n=5) the composite outcome occurred in 20% of patients at 1 year. At baseline, the positive likelihood ratio (LR+) of dd-cfDNA to detect AR by the center pathologist was 3.74 (95% CI 3.01 - 4.64) and core pathologist was 2.59 (95%CI: 1.95 - 3.45). If the composite outcome was included as a true positive, the LR+ of dd-cfDNA improved to 9.82 (95%CI: 7.04, 13.69) and7.63 (95% CI: 5.61, 10.38) at 1-year, respectively. CONCLUSIONS: Pathologists interrater reliability is limited in both ACR and AMR. The positive LR of dd-cfDNA when compared to traditional histopathology is limited, but when longitudinal clinical outcomes are included to assess diagnostic performance, the LR+ improves significantly. The value of dd-cfDNA extends beyond the diagnosis of AR to include other clinically meaningful outcomes for patients after heart transplant.

Evaluating the breadth of nucleic acid-based payloads delivered in lipid nanoparticles to establish fundamental differences in development.

INTRODUCTION: Nucleic acid (NA)-based therapeutics have shown great potential for downregulating or augmenting gene expression, and for promising applications, e.g., protein-replacement therapy and vaccination, a comprehensive understanding of the requirements for their targeted delivery to specific tissues or cells is needed. AREAS COVERED: In this review, we discuss clinical applications of four representative types of NA-based therapeutics, i.e. antisense oligonucleotides, small interfering RNA, messenger RNA, and circular RNA, with a focus on the lipid nanoparticle (LNP) technology used for intracellular delivery. The in vivo fate of LNPs is discussed to improve the understanding of trafficking of nanomedicines at the systemic and cellular levels. In addition, NA-based vaccines are discussed, focusing on targeting antigen-presenting cells and immune activation. EXPERT OPINION: Optimization of delivery systems for NA-based therapeutics is mainly focused on the standard requirements of prolonged systemic circulation and enhancing endosomal escape. Depending on the final destination in specific target tissues or cells, strategies should be adjusted to achieve the desired biodistribution of NA-based payloads. More studies relating to the pharmacokinetics of both cargo and carrier are encouraged, because their in vivo fates may differ, considering the possibility of premature cargo release before reaching the target.

Fluorescence Polarization Assay for Infection Diagnostics: A Review.

Rapid and specific diagnosis is necessary for both the treatment and prevention of infectious diseases. Bacteria and viruses that enter the bloodstream can trigger a strong immune response in infected animals and humans. The fluorescence polarization assay (FPA) is a rapid and accurate method for detecting specific antibodies in the blood that are produced in response to infection. One of the first examples of FPA is the non-competitive test for detecting brucellosis in animals, which was followed by the development of other protocols for detecting various infections. Fluorescently labeled polysaccharides (in the case of brucellosis and salmonellosis) or specific peptides (in the case of tuberculosis and salmonellosis, etc.) can be used as biorecognition elements for detecting infections. The availability of new laboratory equipment and mobile devices for fluorescence polarization measurements outside the laboratory has stimulated the development of new fluorescence polarization assays (FPAs) and the emergence of commercial kits on the market for the detection of brucellosis, tuberculosis, and equine infectious anemia viruses. It has been shown that, in addition to antibodies, the FPA method can detect both viruses and nucleic acids. The development of more specific and sensitive biomarkers is essential for the diagnosis of infections and therapy monitoring. This review summarizes studies published between 2003 and 2023 that focus on the detection of infections using FPA. Furthermore, it demonstrates the potential for using new biorecognition elements (e.g., aptamers, proteins, peptides) and the combined use of FPA with new technologies, such as PCR and CRISPR/Cas12a systems, for detecting various infectious agents.

Lamin chromatin binding is modulated by interactions of different LAP2α domains with lamins and chromatin.

Lamins A and C are components of the lamina at the nuclear periphery and associate with heterochromatin. A distinct, relatively mobile pool of lamin A/C in the nuclear interior associates with euchromatic regions and with lamin-associated polypeptide 2α (LAP2α). Here we show that phosphorylation-dependent impairment of lamin assembly had no effect on its chromatin association, while LAP2α depletion was sufficient to increase chromatin association of lamins. This suggests that complex interactions between LAP2α, chromatin, and lamins regulate lamin chromatin binding. Both the C terminus of LAP2α and its N-terminal LAP2-Emerin-MAN1 (LEM) domain, mediating interaction with lamin A/C indirectly via barrier-to-autointegration factor (BAF), are required for binding to lamins. The N-terminal LEM-like domain of LAP2α, but not its LEM domain, mediates chromatin association of LAP2α and requires LAP2α dimerization via its C terminus. Our data suggest that formation of several LAP2α-, lamin A/C-, and BAF-containing complexes in the nucleoplasm and on chromatin affects lamin chromatin association.

Specifically Editing Cancer Sialoglycans for Enhanced Immunotherapy In Vivo through Aptamer-Enzyme Chimeras.

Immune checkpoints blockade (ICB) therapies have demonstrated remarkable clinical success in treating cancer. However, its objective response rate remains suboptimal because current therapies rely on limited immune checkpoints that failed to cover the multiple immune evasion pathways of cancer. To explore potential ICB strategies, herein, we propose a glycoimmune checkpoint elimination (glycoICE) therapy depending on targeted edition of sialoglycans on tumor cell surface using aptamer-enzyme chimera (ApEC). The ApEC is readily generated via a one-step bioorthogonal procedure, allowing for large-scale and uniform production. The ApEC is able to target and desialylate cancer cells, leading to the elimination of sialoglycan-Siglec axis, which in turn activates immune cells and enhances immunotherapy efficiency. In addition to its remarkable therapeutic efficiency, the ApEC exhibits high tumor selectivity, which helps to avoid side effects caused by indiscriminate desialylation of normal tissues. Furthermore, the ApEC has the potential to be a versatile platform for specifical editing of sialoglycans in different tumor models by adjusting the aptamer sequences targeting associated with specific protein markers. This research not only introduces a novel molecular tool for the effective editing of sialoglycans in complex environments, but also provides valuable insights for advancing DNA-based drugs towards in vivo and clinical applications.

Application of smart-responsive hydrogels in nucleic acid and nucleic acid-based target sensing: A review.

In recent years, nucleic acid-related sensing and detection have become essential in clinical diagnostics, treatment and genotyping, especially in connection with the Human Genome Project and the COVID-19 pandemic. Many traditional nucleic acid-related sensing strategies have been employed in analytical chemistry, including fluorescence, colorimetric and chemiluminescence methods. However, their key limitation is the lack of understanding of the interaction during analysis, particularly at the 3D matrix level close to biological tissue. To address this issue, smart-responsive hydrogels are increasingly used in biosensing due to their hydrophilic and biocompatible properties. By combining smart-responsive hydrogels with traditional nucleic acid-related sensing, biological microenvironments can be mimicked, and targets can be easily accessed and diffused, making them ideal for nucleic acid sensing. This review focuses on utilizing smart-responsive hydrogels for nucleic acid-related sensing and detection, including nucleic acid detection, other nucleic acid-based analyte detection and nucleic acid-related sensing platforms applying nucleic acid as sensing tools in hydrogels. Additionally, the analytical mechanisms of smart-responsive hydrogels with the combination of various detection platforms such as optical and electrochemical techniques are described. The limitations of using smart-responsive hydrogels in nucleic acid-related sensing and proposed possible solutions are also discussed. Lastly, the future challenge of smart-responsive hydrogels in nucleic acid-related sensing is explored. Smart-responsive hydrogels can be used as biomimetic materials to simulate the extracellular matrix, achieve biosensing, and exhibit great potential in nucleic acid-related sensing. They serve as a valuable complement to traditional detection and analytical methods.