Search Results (380)

A straightforward approach is suggested to selectively recognize specific products of loop-mediated isothermal amplification (LAMP) with the Cas12a nuclease without a need for a protospacer adjacent motif (PAM) in the sequence of LAMP amplicons (LAMPlicons). This strategy is based on the presence of single-stranded DNA loops in LAMPlicons and the ability of Cas12a to be trans-activated via the binding of guide RNA (gRNA) to single-stranded DNA in the absence of PAM. The approach feasibility is demonstrated on Clavibacter species—multiple bacterial plant pathogens that cause harmful diseases in agriculturally important plants. For Clavibacter species, the detection sensitivity of the developed PAM-independent LAMP/Cas12a system was determined by that of LAMP. The overall detection selectivity was enhanced by the Cas12a analysis of LAMPlicons. It was shown that the LAMP/Cas12a detection system can be fine-tuned by carefully designing gRNA to selectively distinguish C. sepedonicus from other Clavibacter species based on single nucleotide substitutions in the targeted LAMPlicon loop. The suggested loop-based Cas12a analysis of LAMPlicons was compatible with the format of a single test tube assay with the option of naked-eye detection. The findings broaden the palette of approaches to designing PAM-independent LAMP/Cas12a detection systems with potential for on-site testing. Full article

Chronic infection with the hepatitis B virus (HBV) results in over 1 million deaths annually. Although currently licensed treatments, including pegylated interferon-α and nucleoside/nucleotide analogs, can inhibit viral replication, they rarely eradicate covalently closed circular DNA (cccDNA) reservoirs. Moreover, vaccination does not offer therapeutic benefit to already infected individuals or non-responders. Consequently, chronic infection is maintained by the persistence of cccDNA in infected hepatocytes. For this reason, novel therapeutic strategies that permanently inactivate cccDNA are a priority. Obligate heterodimeric transcription activator-like effector nucleases (TALENs) provide the precise gene-editing needed to disable cccDNA. To develop this strategy using a therapeutically relevant approach, TALEN-encoding mRNA targeting viral core and surface genes was synthesized using in vitro transcription with co-transcriptional capping. TALENs reduced hepatitis B surface antigen (HBsAg) by 80% in a liver-derived mammalian cell culture model of infection. In a stringent HBV transgenic murine model, a single dose of hepatotropic lipid nanoparticle-encapsulated TALEN mRNA lowered HBsAg by 63% and reduced viral particle equivalents by more than 99%, without evidence of toxicity. A surveyor assay demonstrated mean in vivo HBV DNA mutation rates of approximately 16% and 15% for Core and Surface TALENs, respectively. This study presents the first evidence of the therapeutic potential of TALEN-encoding mRNA to inactivate HBV replication permanently. Full article

Genome editing is commonly used in biomedical research. Among the genome editing tools, zinc finger nucleases (ZFNs) are smaller in size than transcription activator-like effector nucleases (TALENs) and CRISPR-Cas9. Therefore, ZFNs are easily packed into a viral vector with limited cargo space. However, ZFNs also consist of left and right monomers, which both need to be expressed in the target cells. When each monomer is expressed separately, two expression cassettes are required, thus increasing the size of the DNA. This is a disadvantage for a viral vector with limited cargo space. We herein showed that T2A-coupled ZF-ND1 monomers were co-expressed from a single expression cassette and that the corresponding ZF-ND1s efficiently cleaved the target DNA sequences. Furthermore, the total amount of transfected plasmid DNA was reduced by half, and genome editing efficiency was equivalent to that of two separate ZF-ND1 monomers. This study provides a promising framework for the development of ZFN applications. Full article

Circular RNAs (circRNAs) have emerged as a transformative class of RNA therapeutics, distinguished by their closed-loop structure conferring nuclease resistance, reduced immunogenicity, and sustained translational activity. While challenges in pharmacokinetic control and manufacturing standardization require resolution, emerging synergies between computational design tools and modular delivery platforms are accelerating clinical translation. In this review, we synthesize recent advances in circRNA therapeutics, with a focused analysis of their stability and immunogenic properties in vaccine and drug development. Notably, key synthesis strategies, delivery platforms, and AI-driven optimization methods enabling scalable production are discussed. Moreover, we summarize preclinical and emerging clinical studies that underscore the potential of circRNA in vaccine development and protein replacement therapies. As both a promising expression vehicle and programmable regulatory molecule, circRNA represents a versatile platform poised to advance next-generation biologics and precision medicine. Full article

For many years, staphylococci have been detected mainly in infections of the skin and soft tissues, organs, bone inflammations, and generalized infections. Thromboembolic diseases have also become a serious plague of our times, which, as it turns out, are closely related to the toxic effects of staphylococci. Staphylococcus aureus, because of the presence of many different kinds of virulence factors, is capable of manipulating the host’s innate and adaptive immune responses. These include toxins and cofactors that activate host zymogens and exoenzymes, as well as superantigens, which are highly inflammatory and cause leukocyte death. Coagulases and staphylokinases can control the host’s coagulation system. Nucleases and proteases inactivate various immune defense and surveillance proteins, including complement components, peptides and antibacterial proteins, and surface receptors that are important for leukocyte chemotaxis. On the other hand, secreted toxins and exoenzymes are proteins that disrupt the endothelial and epithelial barrier as a result of cell lysis and disintegration of linking proteins, which ultimately increases the risk of thromboembolism. In this review, we discuss various virulence factors and substances that may inhibit their activity. Full article

Trypanosoma cruzi is a kinetoplastid parasite and etiological agent of Chagas disease. Given the significant morbidity and mortality rates of this parasitic disease, possible treatment alternatives need to be studied. 3-Bromopyruvate (3-BrPA) is a synthetic analog of pyruvate that was introduced in the early 21st century as an anticancer agent, affecting the proliferation and motility of certain microorganisms. Therefore, this work aims to evaluate the role of 3-BrPA in the energy metabolism, proliferation, and infectivity of T. cruzi, with a primary focus on the mitochondrial state, ATP production, and the key glycolytic pathway enzymes. It was observed that mitochondrial function in 3-BrPA cells was impaired compared to control cells. Accordingly, cells maintained in control conditions have a higher intracellular ATP content than cells maintained with 3-BrPA and higher ecto-phosphatase activity. However, the 3-BrPA reduced ecto-nuclease activity and was capable of hydrolyzing 5′-AMP, ADP, and ATP. When we evaluated two key glycolytic pathway enzymes, glucose kinase (GK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), we observed that 3-BrPA induced higher GAPDH activity but did not alter GK activity. The compensatory energy mechanisms presented in T. cruzi may influence the process of cell metabolism and, consequently, the functional infectious process, suggesting the potential use of 3-BrPA in future clinical applications for Chagas disease. Full article

In recent years, global public health security has encountered significant challenges, with infectious diseases accounting for approximately 25% of global mortality annually. The worldwide pandemic instigated by the novel coronavirus, alongside the persistent threats posed by Ebola, influenza, and multidrug-resistant bacteria, has severely compromised human health, economic development, and social stability. Within this context, the development of rapid and precise pathogen detection technologies has emerged as a critical frontline defense for epidemic prevention and control, serving as a pivotal component in the implementation of the “early detection, early isolation, and early treatment” strategy. The Argonaute (Ago) protein, recognized as a programmable and target-specific activated nuclease, has demonstrated substantial potential in the realm of nucleic acid detection due to its distinctive biological properties, garnering considerable attention. In this study, we delineate the structural characteristics of Ago proteins and elucidate the mechanism underlying their nuclease activity. Furthermore, we review the principles of nucleic acid detection based on Argonaute and provide a comprehensive analysis of recent advancements in related detection systems. Additionally, we compare the advantages of detection based on Argonaute with other detection methodologies. Through a comprehensive analysis, we aim to provide a robust theoretical foundation and an advanced technical reference for the development of new-generation nucleic acid detection platforms with high sensitivity and high specificity. Full article

T40214 (STAT) and its recently investigated analogue STATB are G-quadruplex (G4) forming aptamers characterized by an unusually high percentage of C. The therapeutic potential of T40214 relies on its ability to inhibit the signalling pathway of STAT3, a protein frequently overexpressed in tumor cells. STAT adopts a dimeric 5′-5′ end-stacked quadruplex structure, characterized by parallel strands, three G-tetrads and three propeller-shaped loops formed by a cytidine residue. STATB folds in a very similar structure, apart from an additional cytidine bulge loop. Many studies suggest that thermal stability and topology of G4 can be significantly affected by C methylation, thus resulting in altered interaction of G4-binding proteins with these structures. Considering this, two series of STAT and STATB analogues containing a single 5-methyl-2′-deoxycytidine (mC) residue instead of canonical C nucleotide in the loop have been prepared and investigated by a combination of spectroscopic and electrophoretic techniques. CD, NMR and PAGE data clearly indicate that all derivatives adopt dimeric G4 strictly similar to that assumed by parent aptamers, but with higher stabilities. Furthermore, the resistance to nucleases and the antiproliferative activity of these mC-containing derivatives against HCT116 (human colorectal carcinoma) and T24 (human bladder carcinoma) cell lines have been evaluated. In most of the cases, STAT and STATB derivatives inhibit cell proliferation to different extents, although to a lesser degree than the unmodified parent sequences. All the data highlight the key role of the loops and indicate mC as a useful tool to contribute favorably to the stability of G4-forming aptamers without alteration of their topology, required for the biological activity. Full article

The human gut microbiota is a complex and dynamic ecosystem, recognized for its valuable and wide array of physiological functions. This study investigated the human gut microbiota as a source of enzymes for innovative applications in the biomedicine, bioremediation, and food and feed biotechnological industries by integrating data from combined in silico and in vitro approaches. A total of 93 easily cultivable strains were selected from a bank of isolated microorganisms generated from the gut microbiota of children under different media and conditions. First, genomic data screening and enzyme interrogation of reference genomes corresponding to the selected species were carried out using a custom bioinformatic searching protocol. The extraction and interpretation of encoding enzymes from the genomic taxa results focused on four major phyla (Bacillota, Bacteroidota, Actinomycetota, and Pseudomonadota) and seven genera (Bacillus, Bacteroides, Clostridium, Enterobacter, Enterococcus, Microbacterium, and Staphylococcus) according to their cultivability and biotechnological relevance and interest. A total of 364 enzymes were identified across protein annotations, highlighting amylases, cellulases, inulinases, lipases, proteases, and laccases. Second, phenotypic assays confirmed these main enzymatic activities in 80.6% of 93 isolates. Notable findings included Bacillus species displaying relevant amylase and laccase activity. This study demonstrates the utility of combining genomic annotations with functional assays, offering a robust approach for exploiting gut microbiota enzymes to develop innovative and sustainable biotechnological processes. Moreover, regulatory mechanisms governing enzyme expression in gut resilient microbes are essential steps toward unlocking the full potential of gut microbiota-derived biocatalysts. Full article

Background/Objectives: PAX3 is a transcription factor that drives melanoma progression by promoting cell growth, migration, and survival, while inhibiting cellular terminal differentiation. However, known PAX3 target genes are limited and cannot fully explain the wide impact of PAX3 function. The PAX3 protein can regulate DNA through two separate binding domains, the Paired Domain (PD) and Homeodomain (HD), which bind different DNA motifs. It is not clear if these two domains bind and work together to regulate genes and if they promote all or only a subset of downstream cellular events. Methods: PAX3 direct downstream targets were identified using Cleavage Under Targets & Release Using Nuclease (CUT&RUN) assays in SK-MEL-5 melanoma cells. PAX3-binding genomic regions were identified through MACS2 peak calling, and peaks were categorized based on the presence of PD and/or HD binding sites (or neither) through HOMER motif analysis. The peaks were further characterized as Active, Primed, Poised, Repressed, or Closed based on ATAC-seq data and CUT&RUN for histone Post-Translational Modifications H3K4me1, H3K4me3, H3K27me3, and H3K27Ac. Results: This analysis revealed that most of the PAX3 binding sites in the SK-MEL-5 cell line were primarily through the PD and connected to Active genes. Surprisingly, PAX3 does not commonly act as a repressor in SK-MEL-5 cells. Pathway analysis identified genes involved with transcription, RNA modification, and cell growth. Peaks located in distal enhancer elements were connected to genes involved in neuronal growth, function, and signaling. Conclusions: Our results reveal novel PAX3 regulatory regions and putative genes in a melanoma cell line, with a predominance of PAX3 PD binding on active sites. Full article

The human (h) growth hormone (GH)/placental lactogen (PL) gene family has served as an important model to study tissue-specific expression. The two GH genes (hGH-N/GH1 and GH-V/GH2) and three PL or chorionic somatomammotropin hormone (CSH) genes (hPL-L/CSL1, hPL-A/CSH1 and hPL-B/CSH2) are clustered together at a single locus. Although they share >90% sequence similarity, hGH-N is expressed by somatotrophs of the anterior pituitary while the remaining four hGH/PL genes are expressed by the villous syncytiotrophoblast of the placenta. Efficient pituitary expression depends on a locus control region (LCR) that includes nuclease hypersensitive sites I-V (HS I-V). For activation, data indicate that HS III facilitates the initial access of pituitary-specific transcription factor Pit-1 to the locus, where it is required to bind Pit-1 sites at HS I/II and the hGH-N promoter. This is associated with histone acetylation and tri-methylation modifications that are consistent with active chromatin. However, all five hGH/PL genes share similar nuclease sensitivity in human pituitary chromatin, suggesting similar levels of accessibility and thus potential for transcription. Furthermore, hPL-A and hPL-B promoters contain Pit-1 binding sites, and the hPL-A promoter, like hGH-N, will support expression in transfected pituitary tumor GC cells in culture. These observations suggest the possibility of a transcriptional repressor mechanism that prevents hPL gene expression in the pituitary. P sequences were identified as a candidate. They are located upstream of all four placental hGH/PL genes but not hGH-N, repress hPL-A promoter activity in transfected pituitary GC cells, and bind a forkhead box A1/nuclear factor-1 transcription, which is proposed to act as a repressor complex in human pituitary chromatin. In spite of this, the inability to limit hGH-N expression when tested in transgenic mice brought the role of P sequences in pituitary repression into question. These observations are re-examined here in light of new evidence that the LCR (HS III) interacts with P sequences in the human pituitary. Full article

Nucleases are critical metabolic enzymes expressed by mycoplasmas to acquire nucleic acid precursors from the host for their parasitic existence. Certain nucleases, either membrane-bound or secreted, not only contribute to the growth of mycoplasmas but also serve as key virulence factors due to their unique spatial structures and physiological activity. The pathogenesis includes, but is not limited to, degradation of host DNA and RNA, leading to disruptions of nucleic acid metabolism and the induction of host cell apoptosis; degradation of neutrophil extracellular traps (NETs), allowing escape from neutrophil-mediated killing; and upregulation of inflammatory molecules to modulate the immune response of the host. Understanding the biological functions of nucleases is essential for gaining deeper insights into the virulence and immune evasion strategies of mycoplasmas, which can inform the development of novel approaches for the prevention, diagnosis, and treatment of mycoplasma infections. Full article

Background: Most newly developed anticancer treatments trigger tumor cell death through apoptosis, for which involvement of caspases activity is essential. However, numerous mutations in apoptotic pathways that lead to cancer and favor resistance to apoptosis are known; most are related to caspase-dependent apoptosis pathways and thus have low efficacy. To overcome these limitations, we constructed a novel chimeric protein, GnRH-AIF, using a gonadotropin-releasing hormone (GnRH) analog as a targeting moiety and the apoptosis-inducing factor (AIF) in its cleaved form as a killing moiety, fused at the cDNA level. AIF has a crucial role in the caspase-independent apoptotic pathway. A wide variety of solid tumors overexpress GnRH-receptors (GnRH-R) that are targeted by the new GnRH-AIF chimeric protein. Methods and Results: In this study, we constructed, expressed, and highly purified GnRH-AIF chimeric proteins. We demonstrated the ability of the chimera to enter and specifically and very efficiently kill solid cancer cell lines overexpressing GnRH-R. Most importantly, upon its entry, GnRH-AIFs translocate to the nucleus where it causes DNA fragmentation leading to a direct caspase-independent apoptotic death. As AIFs lack nuclease activity, our findings also emphasize that cell death induced by GnRH-AIF is dependent on the presence of the ENDOG and PPIA proteins, known to participate in the formation of a DNA–degradosome complex. Finally, we demonstrated the high anti-tumor efficacy of the GnRH-AIF ex vivo, in a human, colon cancer organoid model. Conclusions: Our study shows the potential of using a GnRH-AIF chimeric protein as a novel approach to treat solid cancers that overexpress GnRH-R, via a caspase-independent apoptotic pathway. Full article

The Bunyavirales order includes a range of zoonotic viruses, which can cause severe disease in humans. The viral replication machinery is a logical target for the development of direct-acting antivirals. Inhibition of the cap-snatching endonuclease activity of related influenza viruses provides a proof of concept. Using the influenza B virus (IBV) RNA-dependent RNA polymerase complex as a benchmark, we conducted a comparative analysis of endonuclease activities of recombinant full-length bunyaviral L proteins using gel-based assays. The IBV complex demonstrates specific endonucleolytic cleavage and a clear preference for capped substrates. In contrast, severe fever with thrombocytopenia syndrome, Sin Nombre, and Hantaan virus L proteins readily cleave capped and uncapped RNAs to a broader spectrum of RNA fragments. Active site mutants further help to control for the potential of contaminating nucleases, exonuclease activity, and RNA hydrolysis. The influenza cap-snatching inhibitor baloxavir and derivatives have been used to validate this approach. In conclusion, the results of this study demonstrate the importance of assays with single nucleotide resolution and the use of full-length L proteins as a valuable experimental tool to identify selective endonuclease inhibitors. Full article

Nucleic-acid-based therapies have emerged as a pivotal domain within contemporary biomedical science, marked by significant advancements in recent years. These innovative treatments primarily operate through the precise binding of DNA or RNA molecules to discrete target genes, subsequently suppressing the expression of the target proteins. The spectrum of nucleic-acid-based therapies encompasses antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs), etc. Compared to more traditional medicinal approaches, nucleic-acid-based therapies stand out for their highly targeted action on specific genes, as well as their potential for chemical modification to improve resistance to nucleases, ensuring sustained therapeutic activity and mitigating immunogenicity concerns. Nevertheless, these molecules’ limited cellular permeability necessitates the deployment of delivery vectors to enhance their intracellular uptake and stability. As nucleic-acid-based therapies progressively display promising pharmacodynamic profiles, there has been a burgeoning interest in these treatments for applications in clinical research. This review aims to summarize the variety of nucleic acid drugs and their mechanisms, evaluate the present status in research and application, discourse on prospective trends, and potential challenges ahead. These innovative therapeutics are anticipated to assume a pivotal role in the management of a wide array of diseases. Full article