Search Results (108)

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression and have emerged as critical biomarkers in various diseases, including cancer. Their stability in bodily fluids and role as oncogenes or tumor suppressors make them attractive targets for non-invasive diagnostics. However, conventional detection methods, such as Northern blotting, RT-PCR, and microarrays, are limited by low sensitivity, lengthy protocols, and limited specificity. Electrochemical biosensors offer a promising alternative, providing high sensitivity, rapid response times, portability, and cost-effectiveness. These biosensors translate miRNA hybridization events into quantifiable electrochemical signals, often leveraging redox-active labels, mediators, or intercalators. Recent advancements in nanomaterials and signal amplification strategies have further enhanced detection capabilities, enabling sensitive, label-free miRNA quantification. This review provides a comprehensive overview of the recent advances in electrochemical biosensing of miRNAs, emphasizing innovative redox-based detection strategies, probe immobilization techniques, and hybridization modalities. The critical challenges and future perspectives in advancing electrochemical miRNA biosensors toward clinical translation and point-of-care diagnostics are discussed. Full article

With the rapid development of modern molecular biology, microRNA (miRNA) has been demonstrated to be closely associated with the occurrence and development of tumors and holds significant promise as a biomarker for the early detection, diagnosis, and treatment of cancer and other diseases. Therefore, detecting miRNA and analyzing it to determine its biological functions are of great significance for the screening and diagnosis of diseases. However, the intrinsic characteristics of miRNAs, including their low abundance, short sequence lengths, and high family-specific sequence homology, render traditional detection methods such as Northern blot hybridization, microarray use, and reverse transcription quantitative PCR (RT-qPCR) inadequate for meeting the stringent requirements of clinical detection in biological samples, a task requiring accuracy, rapidity, high detection power, specificity, and cost-effectiveness. In recent years, a substantial amount of effort has been put into developing innovative methodologies to address these challenges. In this review, we aim to provide a comprehensive overview of the recent advancements in these methodologies and their applications in clinical biological sample detection for disease diagnosis. Full article

Aquaporin 1 is a membrane water channel protein, which was studied here in spiny dogfish (Squalus acanthias) osmoregulatory tissues using a variety of techniques. The cloning of aquaporin 1 (AQP1) in the spiny dogfish identified a splice variant version of the mRNA/protein (AQP1SV1/AQP1SV1). Polymerase chain reaction (PCR) in a range of tissues showed AQP1 to be expressed at very high levels in the rectal gland with ubiquitous mRNA expression at lower levels in other tissues. Northern blotting showed that AQP1 had a mRNA size of 5.3 kb in kidney total RNA. The level of AQP1 mRNA was significantly lower in the rectal glands of fish acclimated to 120% seawater (SW; vs. 75% SW (p = 0.0007) and 100% SW (p = 0.0025)) but was significantly higher in those fish in the kidney (vs. 100% SW (p = 0.0178)) and intestine (vs. 75% SW (p= 0.0355) and 100% SW (p = 0.0285)). Quantitative PCR determined that AQP1SV1 mRNA levels were also significantly lower in the rectal glands of both 120% (p = 0.0134) and 100% SW (p = 0.0343) fish in comparison to 75% SW-acclimated dogfish. Functional expression in Xenopus oocytes showed that AQP1 exhibited significant apparent membrane water permeability (p = 0.000008–0.0158) across a range of pH values, whereas AQP1SV1 showed no similar permeability. Polyclonal antibodies produced against AQP1 (AQP1 and AQP1/2 antibodies) and AQP1SV1 had bands at the expected sizes of 28 kDa and 24 kDa, respectively, as well as some other banding. The weak AQP1 antibody and the stronger AQP1/2 antibody exhibited staining in the apical membranes of rectal gland secretory tubules, particularly towards the periphery of the gland. In the gill, the AQP1/2 antibody in particular showed staining in secondary-lamellar pavement-cell basal membranes, and in blood vessels and connective tissue in the gill arch. In the spiral valve intestine side wall and valve flap, the AQP1/2 antibody stained muscle tissue and blood vessel walls and, after tyramide signal amplification, showed some staining in the apical membranes of epithelial cells at the ends of the luminal surface of epithelial folds. In the rectum/colon, there was also some muscle and blood vessel staining, but the AQP1 and AQP1/2 antibodies both stained a layer of cells at the base of the surface epithelium. In the kidney convoluted bundle zone, all three antibodies stained bundle sheath membranes to variable extents, and the AQP1/2 antibody also showed staining in the straight bundle zone bundle sheath. In the kidney sinus zone, the AQP1/2 antibody stained the apical membranes of late distal tubule (LDT) nephron loop cells most strongly, with the strongest staining in the middle of the LDT loop and in patches towards the start of the LDT loop. There was also a somewhat less strong staining of segments of the first sinus zone nephron loop, particularly in the intermediate I (IS-I) tubule segment. Some tubules appeared to show no or only low levels of staining. The results suggest that AQP1 plays a role in rectal gland fluid secretion, kidney fluid reabsorption and gill pavement-cell volume regulation and probably a minor role in intestinal/rectal/colon fluid absorption. Full article

This descriptive, observational, cross-sectional study evaluated HTLV-1 and HTLV-2 infections in Ananindeua, northern Brazil. Individuals were screened for anti-HTLV-1/2 using ELISA (Murex HTLV-I + II, DiaSorin). Reactive or indeterminate samples underwent confirmation via Western blot (HTLV Blot 2.4 kit, MP Diagnostics) and/or RT-qPCR. A questionnaire examined behavioral and risk factors for HTLV-1/2 infection. HTLV-positive individuals received counseling, nurse follow-up, and specialized medical care. Among the 228 individuals investigated, 6 (2.7%) were infected with HTLV-1: 4 men (66.67%) and 2 women (33.33%), aged 51–73 years. The only significant risk factor observed was blood transfusion. Additionally, 80 other individuals residing in the municipality of Ananindeua independently visited the laboratory for an HTLV-1/2 diagnosis. Among them, 23 were diagnosed with HTLV-1 infection, and 1 with HTLV-2. Among the 30 positive individuals, 80% were asymptomatic, while 20% exhibited clinical manifestations associated with HTLV infection, including HAM and Sézary syndrome. These results indicate a notable prevalence of HTLV-1 infection in the municipality of Ananindeua emphasizing the significance of diagnosing the infection to assess its prevalence across the country accurately. Full article

Hemerocallis middendorffii is widely used in the landscaping of Northern China for its exceptional ornamental and ecological attributes. It is also the focus of a substantial body of germplasm development and stress tolerance research. However, the absence of an efficient regeneration and genetic transformation system has been a critical barrier to conducting gene function studies on this species. In this research, the aerial parts of seed-derived H. middendorffii plantlets were used as explants, and the callus induction, proliferation, subculture, differentiation, and rooting conditions in the in vitro regeneration process were optimized. A callus induction rate of 95.6% was achieved, with a regeneration rate of 84.4%. Based on this procedure, a simple and effective Agrobacterium-mediated genetic transformation system was preliminarily developed using a hygromycin-based selection system. The system comprised an Agrobacterium tumefaciens culture solution optical density at 600 nm (OD₆₀₀) of 0.6, an acetosyringone concentration of 100 μmol·L⁻¹ in both the A. tumefaciens infection solution and the co-cultivation medium, a sterilization culture with Timentin at 300 mg·L⁻¹, and a selection culture with hygromycin at 9 mg·L⁻¹. Transgenic H. middendorffii T0 rooted plants were produced within a 5-month period, with a transformation rate of 11.9% and positive rate of 32.8%. The regeneration and genetic transformation system established in this study should help advance functional gene research and genetic improvement in H. middendorffii. However, the genetic transformation was only validated in the T0 plants. To confirm stable integration and long-term transgene stability, future research on the phenotypic and molecular characterization of T1 progeny, including segregation analysis and Southern blot verification, will be conducted. Full article

MicroRNAs (miRNAs), which circulate in the serum and plasma, play a role in several biological processes, and their levels in body fluids are associated with the pathogenesis of various diseases, including different types of cancer. For this reason, miRNAs are considered promising candidates as biomarkers for diagnostic purposes, enabling the early detection of pathological onset and monitoring drug responses during therapy. However, current methods for miRNA quantification, such as northern blotting, isothermal amplification, RT-PCR, microarrays, and next-generation sequencing, are limited by their reliance on centralized laboratories, high costs, and the need for specialized personnel. Consequently, the development of sensitive, simple, and one-step analytical techniques for miRNA detection is highly desirable, particularly given the importance of early diagnosis and prompt treatment in cases of cancer. Lateral flow assays (LFAs) are among the most attractive point-of-care (POC) devices for healthcare applications. These systems allow for the rapid and straightforward detection of analytes using low-cost setups that are accessible to a wide audience. This review focuses on LFA-based methods for detecting and quantifying miRNAs associated with the diagnosis of various cancers, with particular emphasis on sensitivity enhancements achieved through the application of different labels and detection systems. Early, non-invasive detection of these diseases through the quantification of tailored biomarkers can significantly reduce mortality, improve survival rates, and lower treatment costs. Full article

Most eukaryotic genes express more than one mature mRNA, defined as transcript variants. This complex phenomenon arises from various mechanisms, such as using alternative transcription start sites and alternative post-transcriptional processing events. The resulting transcript variants can lead to synthesizing proteins that possess distinct functional domains or may even generate noncoding RNAs, each with unique roles in cellular processes. The generation of these transcript variants is not merely a random occurrence; it is cell-type specific and varies with developmental stages, aging processes, or pathogenesis of diseases. This highlights the biological significance of transcript variants in regulating gene expression and their potential impact on cellular functionality. Despite the biological importance, investigating transcript variants has been hampered by challenges associated with detecting their expression. This review article addresses the advancements in molecular techniques in detecting transcript variants. Traditional methods such as RT-PCR and RT-qPCR can easily detect known transcript variants using primers that target unique exons associated with the variants. Other techniques like RACE-PCR and hybridization-based methods, including Northern blotting, RNase protection assays, and microarrays, have also been utilized to detect transcript variants. Nevertheless, RNA sequencing (RNA-Seq) has emerged as a powerful technique for identifying transcript variants, especially those with previously unknown sequences. The effectiveness of RNA sequencing in transcript variant detection depends on the specific sequencing approach and the precision of data analysis. By understanding the strengths and weaknesses of each laboratory technique, researchers can develop more effective strategies for detecting mRNA transcript variants. This ability will be crucial for our comprehensive understanding of gene regulation and the implications of transcript diversity in various biological contexts. Full article

MicroRNA122 (miR-122) is a microRNA that is highly expressed in hepatocytes and has been identified as a prospective therapeutic target and biomarker for liver injury. An expanding body of research has demonstrated that miR-122 is a critical regulator in both the initiation and progression of a wide range of liver diseases. Traditional methods for detecting miR-122 mainly include Northern blotting and qRT-PCR, but they are technically complex and cumbersome, requiring expensive instruments and high technical requirements. In this paper, we present a novel rapid testing method utilizing a lateral flow nucleic acid biosensor (LFNAB) for the sensitive and time-efficient detection of miR-122. This approach offers several advantages, including a high specificity for miR-122, the ability to detect low concentrations of the target molecule, and a significantly reduced testing time compared to conventional detection methods. In this study, a thiol-modified single-stranded detection DNA probe (Det-DNA), a biotinylated single-stranded capture DNA probe (Cap-DNA), and a biotinylated single-stranded control DNA probe (Con-DNA) are used to construct the LFNAB. A gold nanoparticle (AuNP) is a colored tag, which is used to label the Det-DNA probe. The principle of detecting miR-122 is based on dual DNA-miRNA hybridization reactions on the LFNAB to form sandwich-type AuNP-Det-DNA-miR-122-Cap-DNA complexes, which are captured on the test area of LFNAB for visualization and quantification. After systematic optimization of conditions of experiment, the response of LFNAB was highly linear within the scope of 0 pM-100 pM miR-122, and the detection limit in 15 min was 3.90 pM. The use of LFNAB to detect miR-122 in serum and fingertip blood has yielded satisfactory results. This successful application indicates the effectiveness of LFNAB in detecting miR-122 in both serum and fingertip blood samples, showcasing its potential utility in clinical and research settings for assessing miR-122 levels in different biological samples. Full article

Euphorbia uralensis belongs to the family Euphorbiaceae and is widely distributed in northern Xinjiang, making it a characteristic plant of the region in Xinjiang, China. The chemical composition and biological activity of Euphorbia uralensis have not yet been reported, although certain compounds isolated from Euphorbia plants in Xinjiang, China, have demonstrated exceptional multidrug resistance (MDR) reversal. This study aims to investigate the chemical components present in Euphorbia uralensis with the potential to reverse MDR. The aerial parts of Euphorbia uralensis were extracted using organic solvents of varying polarities, resulting in dichloromethane (Fr-E) and petroleum ether (Fr-S) fractions, which exhibited greater MDR reversal activity than the other fractions. The chemical constituents of the Fr-S fraction were analyzed using GC-MS. The chemical components of the Fr-E fraction were isolated and purified using column chromatography. The most effective compounds with MDR reversal activity were screened out, and the mechanism was investigated using molecular docking, molecular dynamics simulations, Western blotting, and rhodamine 123 staining. GC-MS analysis showed that the Fr-S fraction was rich in triterpenes, fatty acids, phenols, and long-chain alkanes, all of which were identified for the first time in Euphorbia uralensis. Among these, palmitic acid was present at a content level of 15.86%. This study notably unveils the discovery of a new compound and 16 previously recorded compounds for the first time in this plant, with the main types identified as steroids, sesquiterpenes, and flavonoids. The isolated compounds were tested for cytotoxicity and MDR reversal activity. The new compounds Euphouralosides A, pubinernoid A, naringenin, and punigratine showed good MDR reversal activity against MCF-7 and MCF-7/ADR cell lines. Punigratine was the most active compound. Moreover, punigratine could stably bind to the ABCB1 protein. Western blot analysis revealed that punigratine did not affect the expression of the ABCB1 protein in cells (p > 0.05). However, following treatment with punigratine (0.16 μM), there was a significant increase the intracellular accumulation of Rh123 in MCF-7/ADR cells (p < 0.05). These findings suggest that punigratine can inhibit the efflux of the ABCB1 protein, thereby overcoming MDR in tumors. This study provides a foundation for further research on the biological activity and medicinal potential of Euphorbia uralensis. Full article

The 18-exon human TKFC gene codes for dual-activity triokinase and FMN cyclase (TKFC) in an ORF, spanning from exon 2 to exon 18. In addition to TKFC-coding transcripts (classified as tkfc type by their intron-17 splice), databases contain evidence for alternative TKFC transcripts, but none of them has been expressed, studied, and reported in the literature. A novel full-ORF transcript was cloned from brain cDNA and sequenced (accession no. DQ344550). It results from an alternative 3′ splice-site in intron 17. The cloned cDNA contains an ORF also spanning from exon 2 to exon 18 of the TKFC gene but with a 56-nt insertion between exons 17 and 18 (classified as tkfc_ins56 type). This insertion introduces an in-frame stop, and the resulting ORF codes for a shorter TKFC variant, which, after expression, is enzymatically inactive. TKFC intron-17 splicing was found to be differentially expressed in human tissues. In a multiple-tissue northern blot using oligonucleotide probes, the liver showed a strong expression of the tkfc-like splice of intron 17, and the heart preferentially expressed the tkfc_ins56-like splice. Through a comparison to global expression data from massive-expression studies of human tissues, it was inferred that the intestine preferentially expresses TKFC transcripts that contain neither of those splices. An analysis of transcript levels quantified by RNA-Seq in the GTEX database revealed an exception to this picture due to the occurrence of a non-coding short transcript with a tkfc-like splice. Altogether, the results support the occurrence of potentially relevant transcript variants of the TKFC gene, differentially expressed in human tissues. (This work is dedicated in memoriam to Professor Antonio Sillero, 1938–2024, for his lifelong mentoring and his pioneering work on triokinase). Full article

This study aimed to describe the prevalence of HTLV-1/2 in quilombola communities in the state of Pará and investigate the possible sociodemographic risk factors associated with the infection, as well as to trace the occurrence of the familial transmission of the virus. A total of 310 individuals living in eight quilombos located in the state of Pará (northern Brazil) were investigated for the presence of anti-HTLV-1/2 antibodies using an enzyme-linked immunosorbent assay (ELISA), and positive samples were confirmed using Western blot and/or real-time quantitative polymerase chain reaction (qPCR). Participants answered a questionnaire about sociodemographic aspects and risk factors for infection. Anti-HTLV-1/2 antibodies were detected in two individuals (one man and one woman), for an overall seroprevalence of 0.65%. Both individuals belonged to the community of São José de Icatú. The search for intrafamilial infection identified two other infected women, which increased the general prevalence of HTLV-1 among the Icatú to 6.25% (4/64). Western blot and qPCR confirmed their HTLV-1 infection, and phylogenetic analysis demonstrated that the isolates were of the cosmopolitan subtype and transcontinental subgroup. Epidemiological investigation of the cases revealed that the three women, at some point in their lives, had a relationship with the infected male individual. HTLV-1 is transmitted silently between individuals in the community of São José de Icatú with a present or past family relationship, stressing the need for screening and laboratory diagnosis to prevent further dissemination of the virus and surveillance of disease emergence. Full article

With advances in technology, diagnostic techniques have become more sophisticated and efficient at detecting biomarkers rapidly. Biomarkers such as microRNA (miRNA), which exhibit exceptional specificity and sensitivity compared with other biomarkers, have garnered particular interest. Composed of 21–24 nucleotides, miRNAs constitute a noncoding RNA group that regulates gene expression, immune system activation, apoptosis, and other cellular processes; hence, they are frequently used as biomarkers for various diseases. This has sparked significant interest regarding the identification of the specific miRNAs implicated in many diseases. Presently, miRNA detection methods include northern blots, reverse transcription-quantitative polymerase chain reaction, and next-generation sequencing. While these methods are all sensitive, they are time-consuming, complex, and expensive, which renders them unsuitable for on-site detection. Surface-enhanced Raman scattering (SERS) can overcome these limitations to enable the sensitive and rapid detection of miRNA. This technique amplifies Raman signals, with signal enhancement levels changing sensitively depending on the distance between the target molecule and substrate. Therefore, this review covers the principle of SERS as a method for detecting miRNAs using nanomaterials, along with examples of nanomaterials and SERS applications. Based on the available literature, SERS is anticipated to enable the convenient, early diagnosis of various diseases, potentially lowering mortality rates. This review could therefore contribute significantly to the advancement of medical and diagnostic technologies. Full article

MicroRNAs (miRNAs) are small, non-coding RNAs that play a crucial role in regulating gene expression. Dysfunction in miRNAs can lead to various diseases, including cancers, neurological disorders, and cardiovascular conditions. To date, approximately 2000 miRNAs have been identified in humans. These small molecules have shown promise as disease biomarkers and potential therapeutic targets. Therefore, identifying miRNA biomarkers for diseases and developing effective miRNA drug delivery systems are essential. Nanotechnology offers promising new approaches to addressing scientific and medical challenges. Traditional miRNA detection methods include next-generation sequencing, microarrays, Northern blotting, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Nanotechnology can serve as an effective alternative to Northern blotting and RT-qPCR for miRNA detection. Moreover, nanomaterials exhibit unique properties that differ from larger counterparts, enabling miRNA therapeutics to more effectively enter target cells, reduce degradation in the bloodstream, and be released in specific tissues or cells. This paper reviews the application of nanotechnology in miRNA detection and drug delivery systems. Given that miRNA therapeutics are still in the developing stages, nanotechnology holds great promise for accelerating miRNA therapeutics development. Full article

Arsenic compounds have been used as therapeutic alternatives for several diseases including cancer. In the following work, we obtained arsenic nanoparticles (AsNPs) produced by an anaerobic bacterium from the Salar de Ascotán, in northern Chile, and evaluated their effects on the human oral squamous carcinoma cell line OECM-1. Resazurin reduction assays were carried out on these cells using 1–100 µM of AsNPs, finding a concentration-dependent reduction in cell viability that was not observed for the non-tumoral gastric mucosa-derived cell line GES-1. To establish if these effects were associated with apoptosis induction, markers like Bcl2, Bax, and cleaved caspase 3 were analyzed via Western blot, executor caspases 3/7 via luminometry, and DNA fragmentation was analyzed by TUNEL assay, using 100 µM cisplatin as a positive control. OECM-1 cells treated with AsNPs showed an induction of both extrinsic and intrinsic apoptotic pathways, which can be explained by a significant decrease in P-Akt/Akt and P-ERK/ERK relative protein ratios, and an increase in both PTEN and p53 mRNA levels and Bit-1 relative protein levels. These results suggest a prospective mechanism of action for AsNPs that involves a potential interaction with extracellular matrix (ECM) components that reduces cell attachment and subsequently triggers anoikis, an anchorage-dependent type of apoptosis. Full article

Background: Aging and obesity are associated with insulin resistance (IR) and low-grade inflammation. Molecularly, IR is characterized by a reduction in glucose uptake and insulin signaling (IRS-1/Akt/AS160 pathway), while inflammation may result from upregulated NF-κB pathway after low Tyr-IκBα phosphorylation. Upregulated phosphatase activity of PTP1B is associated with impaired insulin signaling and increased inflammation. Plasma levels of palmitic acid (PA) are elevated in obesity, triggering inflammation and disruption of insulin signaling. Traditional medicine in Northern Chile uses oral infusions of Lampaya medicinalis Phil. (Verbenaceae) to treat inflammatory conditions. Significant amounts of flavonoids are found in the hydroethanolic extract of Lampaya (HEL), which may account for its biological activity. The aim of this work was to study the effect of HEL and PA on insulin signaling and glucose uptake as well as inflammatory marker expression in human adipocytes. Methods: We studied HEL effects on PA-induced impairment on insulin signaling, glucose uptake and inflammatory marker content in human SW872 adipocytes. HEL cytotoxicity was assessed in adipocytes at different concentrations (0.01 to 10 g/mL). Adipocytes were incubated or not with PA (0.4 mM, 24 h) with or without HEL (2 h pre-incubation), and then stimulated with insulin (10 min, 100 mM) or a vehicle. Phospho-IRS-1, phospho-Akt, phospho-AS160, phospho-NF-κB and phospho-IκBα, as well as protein levels of PTP1B, were assessed using Western blotting, and glucose uptake was evaluated using the 2-NBDG analogue. Results: At the assessed HEL concentrations, no cytotoxic effects were observed. PA decreased insulin-stimulated phospho-Akt and glucose uptake, while co-treatment with HEL increased such markers. PA decreased phospho-IRS-1 and phospho-Tyr-IκBα. On the other hand, incubation with HEL+PA decreased phospho-AS160 and phospho-NF-κB compared with cells treated with PA alone. Conclusion: Our results suggest a beneficial effect of HEL by improving PA-induced impairment on molecular markers of insulin signaling, glucose uptake and inflammation in adipocytes. Further studies are necessary to elucidate whether lampaya may constitute a preventive strategy for people whose circulating PA levels contribute to IR and inflammation during aging and obesity. Full article