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Biosensors May 2024In this study, we report a multiplexed platform for the simultaneous determination of five marine toxins. The proposed biosensor is based on a disposable electrical...
In this study, we report a multiplexed platform for the simultaneous determination of five marine toxins. The proposed biosensor is based on a disposable electrical printed (DEP) microarray composed of eight individually addressable carbon electrodes. The electrodeposition of gold nanoparticles on the carbon surface offers high conductivity and enlarges the electroactive area. The immobilization of thiolated aptamers on the AuNP-decorated carbon electrodes provides a stable, well-orientated and organized binary self-assembled monolayer for sensitive and accurate detection. A simple electrochemical multiplexed aptasensor based on AuNPs was designed to synchronously detect multiple cyanotoxins, namely, microcystin-LR (MC-LR), Cylindrospermopsin (CYL), anatoxin-α, saxitoxin and okadaic acid (OA). The choice of the five toxins was based on their widespread presence and toxicity to aquatic ecosystems and humans. Taking advantage of the conformational change of the aptamers upon target binding, cyanotoxin detection was achieved by monitoring the resulting electron transfer increase by square-wave voltammetry. Under the optimal conditions, the linear range of the proposed aptasensor was estimated to be from 0.018 nM to 200 nM for all the toxins, except for MC-LR where detection was possible within the range of 0.073 to 150 nM. Excellent sensitivity was achieved with the limits of detection of 0.0033, 0.0045, 0.0034, 0.0053 and 0.0048 nM for MC-LR, CYL, anatoxin-α, saxitoxin and OA, respectively. Selectivity studies were performed to show the absence of cross-reactivity between the five analytes. Finally, the application of the multiplexed aptasensor to tap water samples revealed very good agreement with the calibration curves obtained in buffer. This simple and accurate multiplexed platform could open the window for the simultaneous detection of multiple pollutants in different matrices.
Topics: Marine Toxins; Biosensing Techniques; Microcystins; Gold; Aptamers, Nucleotide; Electrochemical Techniques; Saxitoxin; Metal Nanoparticles; Cyanobacteria Toxins; Bacterial Toxins; Uracil; Tropanes; Alkaloids; Okadaic Acid; Electrodes; Limit of Detection
PubMed: 38920572
DOI: 10.3390/bios14060268 -
Iranian Journal of Public Health Mar 2024We aimed to investigate miR-21-5p inhibition effect on lncRNA-XIST expression and apoptosis status of MCF-7 cells.
BACKGROUND
We aimed to investigate miR-21-5p inhibition effect on lncRNA-XIST expression and apoptosis status of MCF-7 cells.
METHODS
The MCF-7 cells were cultured and transfected by the anti-miR-21-5p oligonucleotide and expression of miR-21-5p, lncRNA-XIST, apoptosis-associated genes ( and ) and one miR-21-5p-unrelated lncRNA (BC200) was assessed by RT-qPCR. Furthermore, cell viability checked by MTT assay and apoptosis and cell cycle in transfected cells were detected by flow cytometry. Also, bioinformatics analysis on the transcriptome data confirmed that the lncRNA XIST might have a critical role in breast cancer (BC) cell apoptosis through ceRNAs mechanism and possible regulatory interactions with miR-21-5p.
RESULTS
Expression of miR-21-5p and lncRNA-XIST was significantly down- and up-regulated respectively (<0.05). However, there was no significant change in lncRNA-BC200 expression. Also, the expression of and upraised significantly (<0.05). In transfected cells, MTT and flow cytometry assays reported a highly significant decrease and increase in viability and apoptosis respectively.
CONCLUSION
Inhibition of miR-21-5p resulted in significant upregulation of lncRNA-XIST and apoptosis-associated genes and , which led to the induction of apoptosis in MCF-7 cells. Therefore, more investigations may provide a valuable target for studies on molecular therapies for BC.
PubMed: 38919297
DOI: 10.18502/ijph.v53i3.15154 -
Biosensors & Bioelectronics Oct 2024Single-chain fragment variables (scFvs), composed of variable heavy and light chains joined together by a peptide linker, can be produced using a cost-effective...
Single-chain fragment variables (scFvs), composed of variable heavy and light chains joined together by a peptide linker, can be produced using a cost-effective bacterial expression system, making them promising candidates for pharmaceutical applications. However, a versatile method for monitoring recombinant-protein production has not yet been developed. Herein, we report a novel anti-scFv aptamer-based biosensing system with high specificity and versatility. First, anti-scFv aptamers were screened using the competitive systematic evolution of ligands by exponential enrichment, focusing on a unique scFv-specific peptide linker. We selected two aptamers, P1-12 and P2-63, with K = 2.1 μM or K = 1.6 μM toward anti-human epidermal growth factor receptor (EGFR) scFv, respectively. These two aptamers can selectively bind to scFv but not to anti-EGFR Fv. Furthermore, the selected aptamers recognized various scFvs with different CDRs, such as anti-4-1BB and anti-hemoglobin scFv, indicating that they recognized a unique peptide linker region. An electrochemical sensor for anti-EGFR scFv was developed using anti-scFv aptamers based on square wave voltammetry. Thus, the constructed sensor could monitor anti-EGFR scFv concentrations in the range of 10-500 nM in a diluted medium for bacterial cultivation, which covered the expected concentration range for the recombinant production of scFvs. These achievements promise the realization of continuous monitoring sensors for pharmaceutical scFv, which will enable the real-time and versatile monitoring of large-scale scFv production.
Topics: Aptamers, Nucleotide; Biosensing Techniques; Single-Chain Antibodies; Humans; ErbB Receptors; Recombinant Proteins; SELEX Aptamer Technique; Electrochemical Techniques
PubMed: 38917513
DOI: 10.1016/j.bios.2024.116511 -
Proceedings of the National Academy of... Jul 2024The essential role of U4 snRNP in pre-messenger RNA (mRNA) splicing has been well established. In this study, we utilized an antisense morpholino oligonucleotide (AMO)...
The essential role of U4 snRNP in pre-messenger RNA (mRNA) splicing has been well established. In this study, we utilized an antisense morpholino oligonucleotide (AMO) specifically targeting U4 snRNA to achieve functional knockdown of U4 snRNP in HeLa cells. Our results showed that this knockdown resulted in global intronic premature cleavage and polyadenylation (PCPA) events, comparable to the effects observed with U1 AMO treatment, as demonstrated by mRNA 3'-seq analysis. Furthermore, our study suggested that this may be a common phenomenon in both human and mouse cell lines. Additionally, we showed that U4 AMO treatment disrupted transcription elongation, as evidenced by chromatin immunoprecipitation sequencing (ChIP-seq) analysis for RNAPII. Collectively, our results identified a unique role for U4 snRNP in the inhibition of PCPA and indicated a model wherein splicing intrinsically inhibits intronic cleavage and polyadenylation in the context of cotranscriptional mRNA processing.
Topics: Humans; Polyadenylation; RNA Precursors; HeLa Cells; Mice; Animals; RNA Splicing; Ribonucleoprotein, U4-U6 Small Nuclear; RNA, Messenger; Introns
PubMed: 38917004
DOI: 10.1073/pnas.2406710121 -
Microbiology Spectrum Jun 2024For facilitating tuberculosis (TB) control, we used a whole-genome sequencing (WGS)-based approach to delineate transmission networks in a country with an intermediate...
UNLABELLED
For facilitating tuberculosis (TB) control, we used a whole-genome sequencing (WGS)-based approach to delineate transmission networks in a country with an intermediate burden of TB. A cluster was defined as isolates with identical genotypes, and an outbreak was defined as clustered cases with epidemiological links (epi-links). To refine a cluster predefined using space oligonucleotide typing and mycobacterial interspersed repetitive unit variable tandem repeat typing, we analyzed one pansusceptible TB (C1) and three multidrug-resistant (MDR)-TB (C2-C4) clusters from different scenarios. Pansusceptible TB cluster (C1) consisting of 28 cases had ≤5 single nucleotide polymorphisms (SNPs) difference between their isolates. C1 was a definite outbreak, with cases attending the same junior high school in 2012. Three MDR-TB clusters (C2-C4) with distinct genotypes were identified, each consisting of 12-22 cases. Some of the cases had either ≤5 or ≤15 SNPs difference with clear or probable epi-links. Of note, even though WGS could effectively assist TB contact tracing, we still observed missing epi-links in some cases within the same cluster. Our results showed that thresholds of ≤5 and ≤15 SNPs difference between isolates were used to categorize definite and probable TB transmission, respectively. Furthermore, a higher SNP threshold might be required to define an MDR-TB outbreak. WGS still needs to be combined with classical epidemiological methods for improving outbreak investigations. Importantly, different SNP thresholds have to be applied to define outbreaks.
IMPORTANCE
TB is a chronic disease. Depending on host factors and TB burden, clusters of cases may continue to increase for several years. Conventional genotyping methods overestimate TB transmission, hampering precise detection of outbreaks and comprehensive surveillance. WGS can be used to obtain SNP information of to improve discriminative limitations of conventional methods and to strengthen delineation of transmission networks. It is important to define the country-specific SNP thresholds for investigation of transmission. This study demonstrated the use of thresholds of ≤5 and ≤15 SNPs difference between isolates to categorize definite and probable transmission, respectively. Different SNP thresholds should be applied while a higher cutoff was required to define an MDR-TB outbreak. The utilization of SNP thresholds proves to be crucial for guiding public health interventions, eliminating the need for unnecessary public health actions, and potentially uncovering undisclosed TB transmissions.
PubMed: 38916321
DOI: 10.1128/spectrum.00418-24 -
BioRxiv : the Preprint Server For... Jun 2024Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase capable of template-independent extension of DNA with random nucleotides. TdT's DNA synthesis...
Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase capable of template-independent extension of DNA with random nucleotides. TdT's DNA synthesis ability has found utility in DNA recording, DNA data storage, oligonucleotide synthesis, and nucleic acid labeling, but TdT's intrinsic nucleotide biases limit its versatility in such applications. Here, we describe a multiplexed assay for profiling and engineering the bias and overall activity of TdT variants in high throughput. In our assay, a library of TdTs is encoded next to a CRISPR-Cas9 target site in HEK293T cells. Upon transfection of Cas9 and sgRNA, the target site is cut, allowing TdT to intercept the double strand break and add nucleotides. Each resulting insertion is sequenced alongside the identity of the TdT variant that generated it. Using this assay, 25,623 unique TdT variants, constructed by site-saturation mutagenesis at strategic positions, were profiled. This resulted in the isolation of several altered-bias TdTs that expanded the capabilities of our TdT-based DNA recording system, Cell History Recording by Ordered Insertion (CHYRON), by increasing the information density of recording through an unbiased TdT and achieving dual-channel recording of two distinct inducers (hypoxia and Wnt) through two differently biased TdTs. Select TdT variants were also tested , revealing concordance between each variant's bias and the bias determined from the multiplexed high throughput assay. Overall, our work, and the multiplex assay it features, should support the continued development of TdT-based DNA recorders, applications of TdT, and further study of the biology of TdT.
PubMed: 38915690
DOI: 10.1101/2024.06.11.598561 -
Cell Reports. Physical Science May 2024Recreating tissue environments with precise control over mechanical, biochemical, and cellular organization is essential for next-generation tissue models for drug...
Recreating tissue environments with precise control over mechanical, biochemical, and cellular organization is essential for next-generation tissue models for drug discovery, development studies, and the replication of disease environments. However, controlling these properties at cell-scale lengths remains challenging. Here, we report the development of printing approaches that leverage polyethylene glycol diacrylate (PEGDA) hydrogels containing photocaged oligonucleotides to spatially program material characteristics with non-destructive, non-ultraviolet light. We further integrate this system with a perfusion chamber to allow us to alter the composition of PEGDA hydrogels while retaining common light-activatable photocaged DNAs. We demonstrate that the hydrogels can capture DNA functionalized materials, including cells coated with complementary oligonucleotides with spatial control using biocompatible wavelengths. Overall, these materials open pathways to orthogonal capture of any DNA functionalized materials while not changing the sequences of the DNA.
PubMed: 38911357
DOI: 10.1016/j.xcrp.2024.101922 -
Journal of Dairy Science Jun 2024Due to its beneficial effects on human health, Bifidobacterium is commonly added to milk powder. Accurate quantification of viable Bifidobacterium is essential for...
Rapid and accurate quantification of viable Bifidobacterium cells in milk powder with a propidium monoazide - antibiotic fluorescence in situ hybridization - flow cytometry method.
Due to its beneficial effects on human health, Bifidobacterium is commonly added to milk powder. Accurate quantification of viable Bifidobacterium is essential for assessing the therapeutic efficacy of milk powder. In this study, we introduced a novel propidium monoazide (PMA) - antibiotic fluorescence in situ hybridization (AFISH) - flow cytometry (FCM) method to rapidly and accurately quantify viable Bifidobacterium cells in milk powder. Briefly, Bifidobacterium cells were treated with chloramphenicol (CM) to increase their rRNA content, followed by staining with RNA-binding oligonucleotide probes, based on the AFISH technique. Then, the DNA-binding dye PMA was used to differentiate between viable and non-viable cells. The PMA-AFISH-FCM method, including sample pretreatment, CM treatment, dual staining, and FCM analysis, required around 2 h and was found to be better than the current methods. This is the first study to implement FCM combined with PMA and oligonucleotide probe for detecting Bifidobacterium.
PubMed: 38908696
DOI: 10.3168/jds.2024-24876 -
Cardiovascular Pathology : the Official... Jun 2024The potential of the pericardial space as a therapeutic delivery tool for cardiac fibrosis and heart failure (HF) treatment has yet to be elucidated. Recently, miRNAs... (Review)
Review
The potential of the pericardial space as a therapeutic delivery tool for cardiac fibrosis and heart failure (HF) treatment has yet to be elucidated. Recently, miRNAs and exosomes have been discovered to be present in human pericardial fluid (PF). Novel studies have shown characteristic human PF miRNA compositions associated with cardiac diseases and higher miRNA expressions in PF compared to peripheral blood. Five key studies found differentially expressed miRNAs in HF, angina pectoris, aortic stenosis, ventricular tachycardia, and congenital heart diseases with either atrial fibrillation or sinus rhythm. As miRNA-based therapeutics for cardiac fibrosis and HF showed promising results in several in vivo studies for multiple miRNAs, we hypothesize a potential role of miRNA-based therapeutics delivered through the pericardial cavity. This is underlined by the favorable results of the first phase 1b clinical trial in this emerging field. Presenting the first human miRNA antisense drug trial, inhibition of miR-132 by intravenous administration of a novel antisense oligonucleotide, CDR132L, established efficacy in reducing miR-132 in plasma samples in a dose-dependent manner. We screened the literature, provided an overview of the miRNAs and exosomes present in PF, and drew a connection to those miRNAs previously elucidated in cardiac fibrosis and HF. Further, we speculate about clinical implications and potential delivery methods.
PubMed: 38906439
DOI: 10.1016/j.carpath.2024.107671 -
Mikrochimica Acta Jun 2024Hepatocellular carcinoma (HCC) is the most common liver malignancy and is characterized by increasing incidence and high mortality rates. Current methods for the...
Hepatocellular carcinoma (HCC) is the most common liver malignancy and is characterized by increasing incidence and high mortality rates. Current methods for the screening and diagnosis of HCC exhibit inherent limitations, highlighting the ever-growing need for the development of new methods for the early diagnosis of HCC. The aim of this work was to develop a novel electrochemical aptasensor for the detection of HepG2 cells, a type of circulating tumor cells that can be used as biomarkers for the early detection of HCC. A carbon screen-printed electrode was functionalized with a composite suspension containing graphene oxide, chitosan, and polyaniline nanoparticles to increase the electrode surface and provide anchoring sites for the HepG2 cell-specific aptamer. The aptamer was immobilized on the surface of the functionalized electrode using multipulse amperometry, an innovative technique that significantly reduces the time required for aptamer immobilization. The innovative platform was successfully employed for the first time for the amplification-free detection of HepG2 cells in a linear range from 10 to 200,000 cells/mL, with a limit of detection of 10 cells/mL. The platform demonstrated high selectivity and stability and was successfully used for the detection of HepG2 cells in spiked human serum samples with excellent recoveries.
Topics: Humans; Hep G2 Cells; Aptamers, Nucleotide; Liver Neoplasms; Electrochemical Techniques; Carcinoma, Hepatocellular; Graphite; Biosensing Techniques; Limit of Detection; Aniline Compounds; Electrodes; Chitosan
PubMed: 38904692
DOI: 10.1007/s00604-024-06479-x