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Applied Microbiology and Biotechnology Jun 2024The human gut microbiota refers to a diverse community of microorganisms that symbiotically exist in the human intestinal system. Altered microbial communities have been...
The human gut microbiota refers to a diverse community of microorganisms that symbiotically exist in the human intestinal system. Altered microbial communities have been linked to many human pathologies. However, there is a lack of rapid and efficient methods to assess gut microbiota signatures in practice. To address this, we established an appraisal system containing 45 quantitative real-time polymerase chain reaction (qPCR) assays targeting gut core microbes with high prevalence and/or abundance in the population. Through comparative genomic analysis, we selected novel species-specific genetic markers and primers for 31 of the 45 core microbes with no previously reported specific primers or whose primers needed improvement in specificity. We comprehensively evaluated the performance of the qPCR assays and demonstrated that they showed good sensitivity, selectivity, and quantitative linearity for each target. The limit of detection ranged from 0.1 to 1.0 pg/µL for the genomic DNA of these targets. We also demonstrated the high consistency (Pearson's r = 0.8688, P < 0.0001) between the qPCR method and metagenomics next-generation sequencing (mNGS) method in analyzing the abundance of selected bacteria in 22 human fecal samples. Moreover, we quantified the dynamic changes (over 8 weeks) of these core microbes in 14 individuals using qPCR, and considerable stability was demonstrated in most participants, albeit with significant individual differences. Overall, this study enables the simple and rapid quantification of 45 core microbes in the human gut, providing a promising tool to understand the role of gut core microbiota in human health and disease. KEY POINTS: • A panel of original qPCR assays was developed to quantify human gut core microbes. • The qPCR assays were evaluated and compared with mNGS using real fecal samples. • This method was used to dynamically profile the gut core microbiota in individuals.
Topics: Humans; Real-Time Polymerase Chain Reaction; Gastrointestinal Microbiome; Feces; Bacteria; Metagenomics; High-Throughput Nucleotide Sequencing; Sensitivity and Specificity; DNA Primers; DNA, Bacterial
PubMed: 38922447
DOI: 10.1007/s00253-024-13204-4 -
Mikrochimica Acta Jun 2024The enumeration of circulating tumor cells (CTCs) in peripheral blood plays a crucial role in the early diagnosis, recurrence monitoring, and prognosis assessment of...
The enumeration of circulating tumor cells (CTCs) in peripheral blood plays a crucial role in the early diagnosis, recurrence monitoring, and prognosis assessment of cancer patients. There is a compelling need to develop an efficient technique for the capture and identification of these rare CTCs. However, the exclusive reliance on a single criterion, such as the epithelial cell adhesion molecule (EpCAM) antibody or aptamer, for the specific recognition of epithelial CTCs is not universally suitable for clinical applications, as it usually falls short in identifying EpCAM-negative CTCs. To address this limitation, we propose a straightforward and cost-effective method involving triplex fluorescently labelled aptamers (FAM-EpCAM, Cy5-PTK7, and Texas Red-CSV) to modify FeO-loaded dendritic SiO nanocomposite (dmSiO@FeO/Apt). This multi-recognition-based strategy not only enhanced the efficiency in capturing heterogeneous CTCs, but also facilitated the rapid and accurate identification of CTCs. The capture efficiency of heterogenous CTCs reached up to 93.33%, with a detection limit as low as 5 cells/mL. Notably, the developed dmSiO@FeO/Apt nanoprobe enabled the swift identification of captured cells in just 30 min, relying solely on the fluorescently modified aptamers, which reduced the identification time by approximately 90% compared with the conventional immunocytochemistry (ICC) technique. Finally, these nanoprobe characteristics were validated using blood samples from patients with various types of cancers.
Topics: Humans; Neoplastic Cells, Circulating; Silicon Dioxide; Aptamers, Nucleotide; Nanocomposites; Fluorescent Dyes; Immunomagnetic Separation; Epithelial Cell Adhesion Molecule; Limit of Detection; Cell Line, Tumor; Ferrosoferric Oxide
PubMed: 38922365
DOI: 10.1007/s00604-024-06504-z -
Neurology International Jun 2024Spinal muscular atrophy is a neuromuscular genetic condition associated with progressive muscle weakness and atrophy. Nusinersen is an antisense oligonucleotide therapy...
Spinal muscular atrophy is a neuromuscular genetic condition associated with progressive muscle weakness and atrophy. Nusinersen is an antisense oligonucleotide therapy approved for the treatment of 5q spinal muscular atrophy in pediatric and adult patients. The objective of this clinical case series is to describe the efficacy and safety of nusinersen in treating spinal muscular atrophy in 20 pediatric and 18 adult patients across six treatment centers in Kuwait. Functional motor assessments (Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders, Hammersmith Functional Motor Scale Expanded, and Revised Upper Limb Module) were used to assess changes in motor function following nusinersen treatment. The safety assessment involved clinical monitoring of adverse events. The results demonstrate clinically meaningful or considerable improvement in motor performance for nearly all patients, lasting over 4 years in some cases. A total of 70% of patients in the pediatric cohort and 72% of patients in the adult cohort achieved a clinically meaningful improvement in motor function following nusinersen treatment. Additionally, nusinersen was well-tolerated in both cohorts. These findings add to the growing body of evidence relating to the clinical efficacy and safety of nusinersen.
PubMed: 38921951
DOI: 10.3390/neurolint16030047 -
Current Issues in Molecular Biology May 2024Sexual maturation of Atlantic salmon males is marked by dramatic endocrine changes and rapid growth of the testes, resulting in an increase in the gonad somatic index...
Sexual maturation of Atlantic salmon males is marked by dramatic endocrine changes and rapid growth of the testes, resulting in an increase in the gonad somatic index (GSI). We examined the association of gonadal growth with serum sex steroids, as well as pituitary and testicular gene expression levels, which were assessed with a DNA oligonucleotide microarray. The testes transcriptome was stable in males with a GSI < 0.08% despite the large difference between the smallest and the largest gonads. Fish with a GSI ≥ 0.23% had 7-17 times higher serum levels of five male steroids and a 2-fold increase in progesterone, without a change in cortisol and related steroids. The pituitary transcriptome showed an upregulation of the hormone-coding genes that control reproduction and behavior, and structural rearrangement was indicated by the genes involved in synaptic transmission and the differentiation of neurons. The observed changes in the abundance of testicular transcripts were caused by the regulation of transcription and/or disproportional growth, with a greater increase in the germinative compartment. As these factors could not be separated, the transcriptome results are presented as higher or lower specific activities (HSA and LSA). LSA was observed in 4268 genes, including many genes involved in various immune responses and developmental processes. LSA also included genes with roles in female reproduction, germinal cell maintenance and gonad development, responses to endocrine and neural regulation, and the biosynthesis of sex steroids. Two functional groups prevailed among HSA: structure and activity of the cilia (95 genes) and meiosis (34 genes). The puberty of A. salmon testis is marked by the predominance of spermatogenesis, which displaces other processes; masculinization; and the weakening of external regulation. Results confirmed the known roles of many genes involved in reproduction and pointed to uncharacterized genes that deserve attention as possible regulators of sexual maturation.
PubMed: 38920991
DOI: 10.3390/cimb46060319 -
Biosensors May 2024A fluorogenic aptamer (FA)-based hybridization chain reaction (HCR) could provide a sensitive and label-free signal amplification method for imaging molecules in living...
A fluorogenic aptamer (FA)-based hybridization chain reaction (HCR) could provide a sensitive and label-free signal amplification method for imaging molecules in living cells. However, existing FA-HCR methods usually face some problems, such as a complicated design and significant background leakage, which greatly limit their application. Herein, we developed an FA-centered HCR (FAC-HCR) method based on a remote toehold-mediated strand displacement reaction. Compared to traditional HCRs mediated by four hairpin probes (HPs) and two HPs, the FAC-HCR displayed significantly decreased background leakage and improved sensitivity. Furthermore, the FAC-HCR was used to test a non-nucleic acid target, apurinic/apyrimidinic endonuclease 1 (APE1), an important BER-involved endonuclease. The fluorescence analysis results confirmed that FAC-HCR can reach a detection limit of 0.1174 U/mL. By using the two HPs for FAC-HCR with polyetherimide-based nanoparticles, the activity of APE1 in living cells can be imaged. In summary, this study could provide a new idea to design an FA-based HCR and improve the performance of HCRs in live cell imaging.
Topics: Aptamers, Nucleotide; DNA-(Apurinic or Apyrimidinic Site) Lyase; Humans; Biosensing Techniques; Nucleic Acid Hybridization; Fluorescent Dyes
PubMed: 38920578
DOI: 10.3390/bios14060274 -
Biosensors May 2024Biosensors play an important role in numerous research fields. Quartz crystal microbalances with dissipation monitoring (QCM-Ds) are sensitive devices, and binding...
Biosensors play an important role in numerous research fields. Quartz crystal microbalances with dissipation monitoring (QCM-Ds) are sensitive devices, and binding events can be observed in real-time. In combination with aptamers, they have great potential for selective and label-free detection of various targets. In this study, an alternative surface functionalization for a QCM-D-based aptasensor was developed, which mimics an artificial cell membrane and thus creates a physiologically close environment for the binding of the target to the sensor. Vesicle spreading was used to form a supported lipid bilayer (SLB) of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphethanolamine-N-(cap biotinyl) (biotin-PE). The SLB was then coated with streptavidin followed by applying a biotinylated aptamer against thrombin. SLB formation was investigated in terms of temperature and composition. Temperatures of 25 °C and below led to incomplete SLB formation, whereas a full bilayer was built at higher temperatures. We observed only a small influence of the content of biotinylated lipids in the mixture on the further binding of streptavidin. The functionalization of the sensor surface with the thrombin aptamer and the subsequent thrombin binding were investigated at different concentrations. The sensor could be reconstituted by incubation with a 5 M urea solution, which resulted in the release of the thrombin from the sensor surface. Thereafter, it was possible to rebind thrombin. Thrombin in spiked samples of human serum was successfully detected. The developed system can be easily applied to other target analytes using the desired aptamers.
Topics: Biosensing Techniques; Thrombin; Lipid Bilayers; Quartz Crystal Microbalance Techniques; Aptamers, Nucleotide; Humans; Phosphatidylcholines
PubMed: 38920574
DOI: 10.3390/bios14060270 -
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 -
Annals of the Academy of Medicine,... Feb 2024Ribonucleic acid (RNA) therapeutics hold great potential for the advancement of dermatological treatments due to, among other reasons, the possibility of treating... (Review)
Review
Ribonucleic acid (RNA) therapeutics hold great potential for the advancement of dermatological treatments due to, among other reasons, the possibility of treating previously undruggable targets, high specificity with minimal side effects, and ability to include multiple RNA targets in a single product. Although there have been research relating to RNA therapeutics for decades, there have not been many products translated for clinical use until recently. This may be because of challenges to the application of RNA therapeutics, including the dearth of effective modes of delivery to the target, and rapid degradation of RNA in the human body and environment. This article aims to provide insight on (1) the wide-ranging possibilities of RNA therapeutics in the field of dermatology as well as (2) how key challenges can be addressed, so as to encourage the development of novel dermatological treatments. We also share our experience on how RNA therapeutics have been applied in the management of hypertrophic and keloid scars.
Topics: Humans; Keloid; Cicatrix, Hypertrophic; RNA; Dermatology; Skin Diseases; Genetic Therapy
PubMed: 38920235
DOI: 10.47102/annals-acadmedsg.2023316 -
Nucleic Acid Therapeutics Jun 2024
PubMed: 38920029
DOI: 10.1089/nat.2024.0032 -
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