Inhibition of survivin by 2'-O-methyl phosphorothioate-modified steric-blocking antisense oligonucleotides.

Chemically modified antisense oligonucleotide (ASO) has been established as a successful therapeutic strategy for treating various human diseases. To date, ten ASO drugs, which are capable of either inducing mRNA degradation via RNase H recruitment (fomivirsen, mipomersen, inotersen, volanesorsen and tofersen) or splice modulation (eteplirsen, nusinersen, golodirsen, viltolarsen and casimersen), have been approved by the regulatory agencies for market entry. Nonetheless, none of these approved drugs are prescribed as cancer therapy. Towards this, we have developed steric-blocking ASOs targeting BIRC5 - a well-validated oncogene. Initial screening was performed by transfection of HepG2 cells with seven BIRC5 exon-2 targeting, uniformly 2'-OMe-PS modified ASOs at 400 nM respectively, leading to the identification of two best-performing candidates ASO-2 and ASO-7 in reducing the production of BIRC5 mRNA. Subsequent dose-response assay was conducted via transfection of HepG2 cells by different concentrations (400, 200, 100, 50, 25 nM) of ASO-2 and ASO-7 respectively, showing that both ASOs consistently and efficiently inhibited BIRC5 mRNA expression in a dose-dependent manner. Furthermore, western blot analysis confirmed that ASO-7 could significantly repress survivin production on protein level. Based on our preliminary results, we believe that ASO-7 could be a useful BIRC5 inhibitor for both research purpose and therapeutic development.

Metallic nanostructure-based aptasensors for robust detection of proteins.

There is a significant need for fast, cost-effective, and highly sensitive protein target detection, particularly in the fields of food, environmental monitoring, and healthcare. The integration of high-affinity aptamers with metal-based nanomaterials has played a crucial role in advancing the development of innovative aptasensors tailored for the precise detection of specific proteins. Aptamers offer several advantages over commonly used molecular recognition methods, such as antibodies. Recently, a variety of metal-based aptasensors have been established. These metallic nanomaterials encompass noble metal nanoparticles, metal oxides, metal-carbon nanotubes, carbon quantum dots, graphene-conjugated metallic nanostructures, as well as their nanocomposites, metal-organic frameworks (MOFs), and MXenes. In general, these materials provide enhanced sensitivity through signal amplification and transduction mechanisms. This review primarily focuses on the advancement of aptasensors based on metallic materials for the highly sensitive detection of protein targets, including enzymes and growth factors. Additionally, it sheds light on the challenges encountered in this field and outlines future prospects. We firmly believe that this review will offer a comprehensive overview and fresh insights into metallic nanomaterials-based aptasensors and their capabilities, paving the way for the development of innovative point-of-care (POC) diagnostic devices.

Splice-Modulating Antisense Oligonucleotides as Therapeutics for Inherited Metabolic Diseases.

The last decade (2013-2023) has seen unprecedented successes in the clinical translation of therapeutic antisense oligonucleotides (ASOs). Eight such molecules have been granted marketing approval by the United States Food and Drug Administration (US FDA) during the decade, after the first ASO drug, fomivirsen, was approved much earlier, in 1998. Splice-modulating ASOs have also been developed for the therapy of inborn errors of metabolism (IEMs), due to their ability to redirect aberrant splicing caused by mutations, thus recovering the expression of normal transcripts, and correcting the deficiency of functional proteins. The feasibility of treating IEM patients with splice-switching ASOs has been supported by FDA permission (2018) of the first "N-of-1" study of milasen, an investigational ASO drug for Batten disease. Although for IEM, owing to the rarity of individual disease and/or pathogenic mutation, only a low number of patients may be treated by ASOs that specifically suppress the aberrant splicing pattern of mutant precursor mRNA (pre-mRNA), splice-switching ASOs represent superior individualized molecular therapeutics for IEM. In this work, we first summarize the ASO technology with respect to its mechanisms of action, chemical modifications of nucleotides, and rational design of modified oligonucleotides; following that, we precisely provide a review of the current understanding of developing splice-modulating ASO-based therapeutics for IEM. In the concluding section, we suggest potential ways to improve and/or optimize the development of ASOs targeting IEM.

Aptamer-engineered (nano)materials for theranostic applications.

A diverse array of organic and inorganic materials, including nanomaterials, has been extensively employed in multifunctional biomedical applications. These applications encompass drug/gene delivery, tissue engineering, biosensors, photodynamic and photothermal therapy, and combinatorial sciences. Surface and bulk engineering of these materials, by incorporating biomolecules and aptamers, offers several advantages such as decreased cytotoxicity, improved stability, enhanced selectivity/sensitivity toward specific targets, and expanded multifunctional capabilities. In this comprehensive review, we specifically focus on aptamer-modified engineered materials for diverse biomedical applications. We delve into their mechanisms, advantages, and challenges, and provide an in-depth analysis of relevant literature references. This critical evaluation aims to enhance the scientific community's understanding of this field and inspire new ideas for future research endeavors.

Nanoparticles-assisted aptamer biosensing for the detection of environmental pathogens.

Given the importance of public health, it is crucial to develop quick, targeted, highly sensitive, and accurate technologies to monitor pathogenic microbes in response to the growing concerns of food and environmental safety. Although conventional approaches for microbiological detection are available, they are laborious, and often skill demanding. Therefore, such approaches are incompetent in the on-site or high-throughput assessment of pathogenic microbes. Numerous efforts have been made to develop biosensors that use nucleic acid aptamer as the biorecognition element, which would avoid the abovementioned limitations. Incorporating nanomaterials (NMs) into aptamer-based biosensors (aptasensors) improves their sensitivity and specificity, opening exciting possibilities for various applications, such as bioanalysis of food and environmental samples. Over the last decade, nanomaterial-conjugated aptasensors have seen a steadily rising demand. To this end, the main goal of this study is to demonstrate the novelty in the design of nanomaterial-conjugated aptasensors and how they can be used to detect different pathogenic microbes in water and food. The intent of this paper is to evaluate the cutting-edge techniques that have appeared in nano-aptasensors throughout the past few years, such as manufacturing procedures, analytical credibility, and sensing mechanisms. Additionally, the fundamental performance parameters of aptasensing techniques (such as detection limits, and sensing ranges response) were also used to evaluate their practical applicability. Finally, it is anticipated that this study will inspire innovative ideas and techniques for the construction and use of aptasensors for monitoring pathogenic microorganisms in food, drinks, recreational water, and wastewater.

Alternative polyadenylation reprogramming of MORC2 induced by NUDT21 loss promotes KIRC carcinogenesis.

Alternative polyadenylation (APA), a posttranscriptional mechanism of gene expression via determination of 3'UTR length, has an emerging role in carcinogenesis. Although abundant APA reprogramming is found in kidney renal clear cell carcinoma (KIRC), which is one of the major malignancies, whether APA functions in KIRC remains unknown. Herein, we found that chromatin modifier MORC2 gained oncogenic potential in KIRC among the genes with APA reprogramming, and moreover, its oncogenic potential was enhanced by 3'UTR shortening through stabilization of MORC2 mRNA. MORC2 was found to function in KIRC by downregulating tumor suppressor DAPK1 via DNA methylation. Mechanistically, MORC2 recruited DNMT3A to facilitate hypermethylation of the DAPK1 promoter, which was strengthened by 3'UTR shortening of MORC2. Furthermore, loss of APA regulator NUDT21, which was induced by DNMT3B-mediated promoter methylation, was identified as responsible for 3'UTR shortening of MORC2 in KIRC. Additionally, NUDT21 was confirmed to act as a tumor suppressor mainly depending on downregulation of MORC2. Finally, we designed an antisense oligonucleotide (ASO) to enhance NUDT21 expression and validated its antitumor effect in vivo and in vitro. This study uncovers the DNMT3B/NUDT21/APA/MORC2/DAPK1 regulatory axis in KIRC, disclosing the role of APA in KIRC and the crosstalk between DNA methylation and APA.

Improving compliance with swallowing exercise to decrease radiotherapy-related dysphagia in patients with head and neck cancer.

Objective: Dysphagia, one of the most common complications in head and neck cancer (HNC) treated with radiotherapy, can severely affect patients' quality of life. Currently, because no "gold standard" treatment exists, swallowing exercise remains the main rehabilitation strategy for dysphagia. However, patients' compliance with long-term swallowing exercise is only 40%, thus, greatly compromising outcomes. This article aims to analyze thefactors influencing swallowing exercise compliance in patients with HNC and explains strategies developed to date for improved rehabilitation outcomes. Methods: Research studies published between 2005 and 2022 were retrieved from seven databases: PubMed, Cochrane Library, Embase, CINAHL, CNKI, Wan Fang Database, and VIP Database, and 21 articles were shortlisted and systematically reviewed. Results: The swallowing exercise compliance in patients with HNC undergoing radiotherapy was affected by multiple factors, including socio-demographic factors, illness-associated factors, treatment-associated factors, and psychosocial factors. Regarding the interventions, current strategies mainly address psychosocial issues via developing various education programs. Conclusions: Different factors influencing swallowing exercise compliance are important and should be observed. Measures including developing multidisciplinary teams, applying innovative equipment, refining the intervention procedure, and applying systematic theory frameworks should be performed to achieve better outcomes of compliance interventions.

Evaluation of Chemically Modified Nucleic Acid Analogues for Splice Switching Application.

In recent years, several splice switching antisense oligonucleotide (ASO)-based therapeutics have gained significant interest, and several candidates received approval for clinical use for treating rare diseases, in particular, Duchenne muscular dystrophy and spinal muscular atrophy. These ASOs are fully modified; in other words, they are composed of chemically modified nucleic acid analogues instead of natural RNA oligomers. This has significantly improved drug-like properties of these ASOs in terms of efficacy, stability, pharmacokinetics, and safety. Although chemical modifications of oligonucleotides have been discussed previously for numerous applications including nucleic acid aptamers, small interfering RNA, DNAzyme, and ASO, to the best of our knowledge, none of them have solely focused on the analogues that have been utilized for splice switching applications. To this end, we present here a comprehensive review of different modified nucleic acid analogues that have been explored for developing splice switching ASOs. In addition to the antisense chemistry, we also endeavor to provide a brief historical overview of the approved spice switching ASO drugs, including a list of drugs that have entered human clinical trials. We hope this work will inspire further investigations into expanding the potential of novel nucleic acid analogues for constructing splice switching ASOs.

Alternative polyadenylation writer CSTF2 forms a positive loop with FGF2 to promote tubular epithelial-mesenchymal transition and renal fibrosis.

Effective therapies for renal fibrosis, the common endpoint for most kidney diseases, are lacking. We previously reported that alternative polyadenylation (APA) drives transition from acute kidney injury to chronic kidney disease, suggesting a potential role for APA in renal fibrogenesis. Here, we found that among canonical APA writers, CSTF2 expression was upregulated in tubular epithelial cells (TEC) of fibrotic kidneys. CSTF2 was also identified as a TGF-ß-inducible pro-fibrotic gene. Further analysis revealed that CSTF2 promoted epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) overproduction in TEC by inducing 3'UTR shortening and upregulation of the expression of basic fibroblast growth factor 2 (FGF2). Additionally, 3'UTR shortening stabilised FGF2 mRNA through miRNA evasion. Interestingly, FGF2 enhanced CSTF2 expression, leading to the forming of a CSTF2-FGF2 positive loop in TEC. Furthermore, CSTF2 knockdown alleviated unilateral ureteral obstruction-induced renal fibrosis in vivo. Finally, we developed a CSTF2-targeted antisense oligonucleotide (ASO) and validated its effectiveness in vitro. These results indicate that the expression of the APA writer, CSTF2, is upregulated by TGF-ß and CSTF2 facilitates TGF-ß-induced FGF2 overexpression, forming a TGF-ß-CSTF2-FGF2 pro-fibrotic axis in TEC. CSTF2 is a potentially promising target for renal fibrosis that does not directly disrupt TGF-ß.

Cyclometalated Ru(II)-isoquinoline complexes overcome cisplatin resistance of A549/DDP cells by downregulation of Nrf2 via Akt/GSK-3ß/Fyn pathway.

Both ruthenium (Ru) and isoquinoline (IQ) compounds are regarded as potential anticancer drug candidates. Here, we report the synthesis and characterization of three novel cyclometalated Ru(II)-isoquinoline complexes: RuIQ-3, RuIQ-4, and RuIQ-5, and evaluation of their in vitro cytotoxicities against a panel of cell lines including A549/DDP, a cisplatin-resistant human lung cancer cell line. A549/DDP 3D multicellular tumor spheroids (MCTSs) were also used to detect the drug resistance reversal effect of Ru(II)-IQ complexes. Our results indicated that the cytotoxic activities against cancer cells of Ru(II)-IQ complexes, especially RuIQ-5, were superior compared with cisplatin. In addition, RuIQ-5 exhibited low toxicity towards both normal HBE cells in vitro and zebrafish embryos in vivo. Further investigation on cellular mechanism of action indicated that after absorption by A549/DDP cells, RuIQ-5 was mainly distributed in the nucleus, which is different from cisplatin. Besides, RuIQ-5 could induce apoptosis through mitochondrial dysfunction, reactive oxygen species (ROS) accumulation, ROS-mediated DNA damage, and cycle arrest at both S and G2/M phases. Moreover, RuIQ-5 could inhibit the overexpression of Nrf2 through regulation of Akt/GSK-3ß/Fyn signaling pathway and hindering the nuclear translocation of Nrf2. Based on these findings, we firmly believe that the studied Ru(II)-IQ complexes hold great promise as anticancer therapeutics with high effectiveness and low toxicity.

Antisense Oligonucleotide-Mediated Splice Switching: Potential Therapeutic Approach for Cancer Mitigation.

Splicing is an essential process wherein precursor messenger RNA (pre-mRNA) is reshaped into mature mRNA. In alternative splicing, exons of any pre-mRNA get rearranged to form mRNA variants and subsequently protein isoforms, which are distinct both by structure and function. On the other hand, aberrant splicing is the cause of many disorders, including cancer. In the past few decades, developments in the understanding of the underlying biological basis for cancer progression and therapeutic resistance have identified many oncogenes as well as carcinogenic splice variants of essential genes. These transcripts are involved in various cellular processes, such as apoptosis, cell signaling and proliferation. Strategies to inhibit these carcinogenic isoforms at the mRNA level are promising. Antisense oligonucleotides (AOs) have been developed to inhibit the production of alternatively spliced carcinogenic isoforms through splice modulation or mRNA degradation. AOs can also be used to induce splice switching, where the expression of an oncogenic protein can be inhibited by the induction of a premature stop codon. In general, AOs are modified chemically to increase their stability and binding affinity. One of the major concerns with AOs is efficient delivery. Strategies for the delivery of AOs are constantly being evolved to facilitate the entry of AOs into cells. In this review, the different chemical modifications employed and delivery strategies applied are discussed. In addition to that various AOs in clinical trials and their efficacy are discussed herein with a focus on six distinct studies that use AO-mediated exon skipping as a therapeutic strategy to combat cancer.

Cyclometalated Ru(II) ß-carboline complexes induce cell cycle arrest and apoptosis in human HeLa cervical cancer cells via suppressing ERK and Akt signaling.

Two new cyclometalated Ru(II)-ß-carboline complexes, [Ru(dmb)2(Cl-Ph-ßC)](PF6) (dmb = 4,4'-dimethyl-2,2'-bipyridine; Cl-Ph-ßC = Cl-phenyl-9H-pyrido[3,4-b]indole; RußC-3) and [Ru(bpy)2(Cl-Ph-ßC)](PF6) (bpy = 2,2'-bipyridine; RußC-4) were synthesized and characterized. The Ru(II) complexes display high cytotoxicity against HeLa cells, the stabilized human cervical cancer cell, with IC50 values of 3.2 ± 0.4 µM (RußC-3) and 4.1 ± 0.6 µM (RußC-4), which were considerably lower than that of non-cyclometalated Ru(II)-ß-carboline complex [Ru(bpy)2(1-Py-ßC)] (PF6)2 (61.2 ± 3.9 µM) by 19- and 15-folds, respectively. The mechanism studies indicated that both Ru(II) complexes could significantly inhibit HeLa cell migration and invasion, and effectively induce G0/G1 cell cycle arrest. The new Ru(II) complexes could also trigger apoptosis through activating caspase-3 and poly (ADP-ribose) polymerase (PARP), increasing the Bax/Bcl-2 ratio, enhancing reactive oxygen species (ROS) generation, decreasing mitochondrial membrane potential (MMP), and inducing cytochrome c release from mitochondria. Further research revealed that RußC-3 could deactivate the ERK/Akt signaling pathway thus inhibiting HeLa cell invasion and migration, and inducing apoptosis. In addition, RußC-3-induced apoptosis in HeLa cells was closely associated with the increase of intracellular ROS levels, which may act as upstream factors to regulate ERK and Akt pathways. More importantly, RußC-3 exhibited low toxicity on both normal BEAS-2B cells in vitro and zebrafish embryos in vivo. Consequently, the developed Ru(II) complexes have great potential on developing novel low-toxic anticancer drugs.

Deviations in the gut microbiota of neonates affected by maternal group B Streptococcus colonization.

BACKGROUND: Group B Streptococcus (GBS) infection is the leading cause of septicemia, meningitis, and pneumonia in neonates. Aberrant gut colonization in early life may predispose children to various diseases in adulthood. However, the associations between gut microbial changes and GBS colonization is still unclear. RESULTS: The composition and diversity of meconium microbiota in GBS group were similar to that of healthy controls. However, we identified several specific taxa that were differentially abundant between the two groups (linear discriminant analysis (LDA) effect size (LEfSe): p < 0.05, LDA > 2.0). Particularly, the relative abundance of Lactobacillus paracasei was significantly reduced, indicating a role in GBS colonization. CONCLUSIONS: Our study presented a series of bacterial species colonized by GBS, thus providing novel evidence in support of initial intestinal microbiota dysbiosis in the neonates with mother's GBS colonization.

Development of nucleic acid aptamer-based lateral flow assays: A robust platform for cost-effective point-of-care diagnosis.

Lateral flow assay (LFA) has made a paradigm shift in the in vitro diagnosis field due to its rapid turnaround time, ease of operation and exceptional affordability. Currently used LFAs predominantly use antibodies. However, the high inter-batch variations, error margin and storage requirements of the conventional antibody-based LFAs significantly impede its applications. The recent progress in aptamer technology provides an opportunity to combine the potential of aptamer and LFA towards building a promising platform for highly efficient point-of-care device development. Over the past decades, different forms of aptamer-based LFAs have been introduced for broad applications ranging from disease diagnosis, agricultural industry to environmental sciences, especially for the detection of antibody-inaccessible small molecules such as toxins and heavy metals. But commercial aptamer-based LFAs are still not used widely compared with antibodies. In this work, by analysing the key issues of aptamer-based LFA design, including immobilization strategies, signalling methods, and target capturing approaches, we provide a comprehensive overview about aptamer-based LFA design strategies to facilitate researchers to develop optimised aptamer-based LFAs.

Antisense Oligonucleotides as Potential Therapeutics for Type 2 Diabetes.

Type 2 diabetes (T2D) is a chronic metabolic disorder characterized by persistent hyperglycemia resulting from inefficient signaling and insufficient production of insulin. Conventional management of T2D has largely relied on small molecule-based oral hypoglycemic medicines, which do not halt the progression of the disease due to limited efficacy and induce adverse effects as well. To this end, antisense oligonucleotide has attracted immense attention in developing antidiabetic agents because of their ability to downregulate the expression of disease-causing genes at the RNA and protein level. To date, seven antisense agents have been approved by the United States Food and Drug Administration for therapies of a variety of human maladies, including genetic disorders. Herein, we provide a comprehensive review of antisense molecules developed for suppressing the causative genes believed to be responsible for insulin resistance and hyperglycemia toward preventing and treating T2D.

Therapeutically Significant MicroRNAs in Primary and Metastatic Brain Malignancies.

Brain cancer is one among the rare cancers with high mortality rate that affects both children and adults. The most aggressive form of primary brain tumor is glioblastoma. Secondary brain tumors most commonly metastasize from primary cancers of lung, breast, or melanoma. The five-year survival of primary and secondary brain tumors is 34% and 2.4%, respectively. Owing to poor prognosis, tumor heterogeneity, increased tumor relapse, and resistance to therapies, brain cancers have high mortality and poor survival rates compared to other cancers. Early diagnosis, effective targeted treatments, and improved prognosis have the potential to increase the survival rate of patients with primary and secondary brain malignancies. MicroRNAs (miRNAs) are short noncoding RNAs of approximately 18-22 nucleotides that play a significant role in the regulation of multiple genes. With growing interest in the development of miRNA-based therapeutics, it is crucial to understand the differential role of these miRNAs in the given cancer scenario. This review focuses on the differential expression of ten miRNAs (miR-145, miR-31, miR-451, miR-19a, miR-143, miR-125b, miR-328, miR-210, miR-146a, and miR-126) in glioblastoma and brain metastasis. These miRNAs are highly dysregulated in both primary and metastatic brain tumors, which necessitates a better understanding of their role in these cancers. In the context of the tumor microenvironment and the expression of different genes, these miRNAs possess both oncogenic and/or tumor-suppressive roles within the same cancer.

Development of Novel Chemically-Modified Nucleic Acid Molecules for Efficient Inhibition of Human MAPT Gene Expression.

The hyperphosphorylation of the microtubule-associated protein tau (MAPT) has been implicated in various neurological diseases, including Alzheimer's disease. It has been hypothesized that the reduction of MAPT would result in depolymerizing neurofibrillary tangles and could be a potential strategy for the treatment of Alzheimer's disease and other tauopathies. In this study, we report the development of novel DNAzymes and splice-modulating antisense oligonucleotides (AOs) for the efficient inhibition of MAPT. We designed and synthesized a range of DNAzymes and 2'-O-methyl (2'-OMe)-modified AOs on a phosphorothioate (PS) backbone targeting various exons across the MAPT gene transcript. Our results demonstrated that RNV563, an arm-loop-arm-type DNAzyme targeting exon 13, and an AO candidate AO4, targeting exon 4, efficiently downregulated MAPT RNA expression by 58% and 96%, respectively. In addition, AO4 also reduced the MAPT protein level by 74%. In line with our results, we believe that AO4 could be used as a potential therapeutic molecule for Alzheimer's disease and other tauopathies.

Author Correction: Systematic evaluation of 2'-Fluoro modified chimeric antisense oligonucleotide-mediated exon skipping in vitro.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Progress, opportunity, and perspective on exosome isolation - efforts for efficient exosome-based theranostics.

Exosomes are small extracellular vesicles with diameters of 30-150 nm. In both physiological and pathological conditions, nearly all types of cells can release exosomes, which play important roles in cell communication and epigenetic regulation by transporting crucial protein and genetic materials such as miRNA, mRNA, and DNA. Consequently, exosome-based disease diagnosis and therapeutic methods have been intensively investigated. However, as in any natural science field, the in-depth investigation of exosomes relies heavily on technological advances. Historically, the two main technical hindrances that have restricted the basic and applied researches of exosomes include, first, how to simplify the extraction and improve the yield of exosomes and, second, how to effectively distinguish exosomes from other extracellular vesicles, especially functional microvesicles. Over the past few decades, although a standardized exosome isolation method has still not become available, a number of techniques have been established through exploration of the biochemical and physicochemical features of exosomes. In this work, by comprehensively analyzing the progresses in exosome separation strategies, we provide a panoramic view of current exosome isolation techniques, providing perspectives toward the development of novel approaches for high-efficient exosome isolation from various types of biological matrices. In addition, from the perspective of exosome-based diagnosis and therapeutics, we emphasize the issue of quantitative exosome and microvesicle separation.

A systematic investigation of key factors of nucleic acid precipitation toward optimized DNA/RNA isolation.

Nucleic acid precipitation is important for virtually all molecular biology investigations. However, despite its crucial role, a systematic study of the influence factors of nucleic acid precipitation has not been reported. In the present work, via rational experimental design, key factors of nucleic acid precipitation, including the type of nucleic acid, temperature and time of incubation, speed and time of centrifugation, volume ratio of ethanol/isopropanol to nucleic acid solution, type of cation-containing salt solution and type of coprecipitator, were comprehensively evaluated in an attempt to maximize the efficiency of nucleic acid precipitation. Our results indicate that the optimal conditions of each influence factor of nucleic acid precipitation may vary in accordance with the chemistry, structure and length of nucleic acids.