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Analytical Methods : Advancing Methods... May 2024In this work, a sensitive colorimetric bioassay method based on a poly(adenine) aptamer (polyA apt) and gold nanoparticles (AuNPs) was developed for the determination of...
In this work, a sensitive colorimetric bioassay method based on a poly(adenine) aptamer (polyA apt) and gold nanoparticles (AuNPs) was developed for the determination of aflatoxin B1 (AFB1). The polyA apt, adsorbed on the AuNPs, especially can bind to the analyte while deterring non-specific interactions. This nano aptasensor uses cationic polymer poly(diallyl dimethyl ammonium chloride) (PDDA), as an aggregating agent, to aggregate gold nanoparticles. PolyA apt-decorated gold nanoparticles (AuNPs/polyA apt) show resistance to PDDA-induced aggregation and maintains their dispersed state (red color) with the optical absorbance signal at = 520 nm. However, in the presence of AFB1 in the assay solution, the specific aptamer reacts with high affinity and folds into its three-dimensional form. Aggregation of AuNPs induced by PDDA caused their optical signal shift to = 620 nm (blue color). AFB1 concentration in the bioassay solution determines the amount of optical signal shift. Therefore, optical density ratio in two wavelengths () can be used as a sturdy colorimetric signal to detect the concentration of aflatoxin B1. AFB1 was linearly detected between 0.5 and 20 ng mL, with a detection limit of 0.09 ng mL (S/N = 3). The fabricated aptasensor was applied to the detection of AFB1 in real corn samples.
Topics: Aflatoxin B1; Gold; Colorimetry; Zea mays; Metal Nanoparticles; Aptamers, Nucleotide; Biosensing Techniques; Poly A; Limit of Detection; Food Contamination; Quaternary Ammonium Compounds; Polyethylenes
PubMed: 38682263
DOI: 10.1039/d4ay00605d -
3 Biotech May 2024Chilli veinal mottle virus (ChiVMV) is a potyvirus known to cause havoc in many solanaceous crops. Samples from tomato plants exhibiting typical mosaic and mottling...
Chilli veinal mottle virus (ChiVMV) is a potyvirus known to cause havoc in many solanaceous crops. Samples from tomato plants exhibiting typical mosaic and mottling symptoms in two locations from farmers' fields were collected and tested using DAC ELISA for the presence of ChiVMV and other viruses known to infect tomato. ChiVMV Gauribidanur isolate from infected tomato was mechanically inoculated to ana, chilli, and tomato plants which exhibited systemic mosaic and mottling symptoms 10 days post-inoculation. This results were further confirmed by RT-PCR and DAC ELISA using CP gene-specific primers and ChiVMV antisera, respectively. Transmission electron microscopy revealed the presence of long filamentous particles (800 × 11 nm) resembling viruses in the family. The complete genome of ChiVMV comprised 9716 nucleotides except for poly A tail, with a predicted open reading frame spanning 9270 nucleotides encoding polyproteins of 3089 amino acids. Comparative analysis revealed that ChiVMV-tomato isolates reported across the world shared maximum nucleotide identity (93-96.7%) with chilli isolates from India and Pakistan. These results were well supported by sequence demarcation analysis. Further, the Neibhor-Net network analysis of the complete genome of ChiVMV-tomato, along with other host isolates, formed a reticular network phylogenetic tree suggesting recombination events. Subsequently, RDP5 detected intra-specific recombination breakpoints at the positions 1656-5666 nucleotides with major parent ChiVMV (MN508960) Uravakonda and minor parent ChiVMV (MN508956) with a significant average value of 1.905 × 10. The LAMP assay using ChiVMV-specific primers resulted in ladder-like amplified products on electrophoresed gel and a distinct red colour pattern with hydroxy naphthalene blue, indicating a positive reaction for the presence of ChiVMV in infected tomato samples. To validate LAMP-designed primers, RNA extracted from ChiVMV-infected tomato, chilli, datura, and tobacco samples were subjected to LAMP assay and it accurately detected the presence of ChiVMV in infected plant samples. Overall, this study provides holistic information of ChiVMV infecting tomato, spanning diagnosis, transmission, genetic characterization, and detection of recombination events, which collectively contribute to effective disease management, crop protection, and informed decision-making in agricultural practices.
PubMed: 38682094
DOI: 10.1007/s13205-024-03984-x -
Biomacromolecules May 2024Studies have shown that poly(adenine) DNA and RNA strands protonate at a low pH to form self-associating duplexes; however, the nanoscopic morphology of these structures...
Studies have shown that poly(adenine) DNA and RNA strands protonate at a low pH to form self-associating duplexes; however, the nanoscopic morphology of these structures is unclear. Here, we use Transition Electron Microscopy (TEM), Atomic Force Microscopy (AFM), dynamic light scattering (DLS), and fluorescence spectroscopy to show that both ribose identity (DNA or RNA) and assembly conditions (thermal or room-temperature annealing) dictate unique hierarchical structures for poly(adenine) sequences at a low pH. We show that while the thermodynamic product of protonating poly(adenine) DNA is a discrete dimer of two DNA strands, the kinetic product is a supramolecular polymer that branches and aggregates to form micron-diameter superstructures. In contrast, we find that protonated poly(A) RNA polymerizes into micrometer-length, twisted fibers under the same conditions. These divergent hierarchical morphologies highlight the amplification of subtle chemical differences between RNA and DNA into unique nanoscale behaviors. With the use of poly(adenine) strands spanning vaccine technologies, sensing, and dynamic biotechnology, understanding and controlling the underlying assembly pathways of these structures are critical to developing robust, programmable nanotechnologies.
Topics: RNA; DNA; Poly A; Protons; Polymers; Microscopy, Atomic Force; Hydrogen-Ion Concentration
PubMed: 38651279
DOI: 10.1021/acs.biomac.4c00271 -
Biochimica Et Biophysica Acta.... Jun 2024Trophoblast cell dysfunction is one of the important factors leading to preeclampsia (PE). Cytoplasmic polyadenylation element-binding 2 (CPEB2) has been found to be...
AIMS
Trophoblast cell dysfunction is one of the important factors leading to preeclampsia (PE). Cytoplasmic polyadenylation element-binding 2 (CPEB2) has been found to be differentially expressed in PE patients, but whether it mediates PE process by regulating trophoblast cell function is unclear.
METHODS
The expression of CPEB2 and somatostatin receptor 3 (SSTR3) was detected by quantitative real-time PCR, Western blot (WB) and immunofluorescence staining. Cell functions were analyzed by CCK-8 assay, EdU assay, flow cytometry and transwell assay. Epithelial-mesenchymal transition (EMT)-related protein levels were detected by WB. The interaction of CPEB2 and SSTR3 was confirmed by RIP assay, dual-luciferase reporter assay and PCR poly(A) tail assay. Animal experiments were performed to explore the effect of CPEB2 on PE progression in vivo, and the placental tissues of rat were used for H&E staining, immunohistochemical staining and TUNEL staining.
RESULTS
CPEB2 was lowly expressed in PE patients. CPEB2 upregulation accelerated trophoblast cell proliferation, migration, invasion and EMT, while its knockdown had an opposite effect. CPEB2 bound to the CPE site in the 3'-UTR of SSTR3 mRNA to suppress SSTR3 translation through reducing poly(A) tails. Besides, SSTR3 overexpression suppressed trophoblast cell proliferation, migration, invasion and EMT, while its silencing accelerated trophoblast cell functions. However, these effects could be reversed by CPEB2 upregulation and knockdown, respectively. In vivo experiments, CPEB2 overexpression relieved histopathologic changes, inhibited apoptosis, promoted proliferation and enhanced EMT in the placenta of PE rat by decreasing SSTR3 expression.
CONCLUSION
CPEB2 inhibited PE progression, which promoted trophoblast cell functions by inhibiting SSTR3 translation through polyadenylation.
Topics: Pregnancy; Humans; Pre-Eclampsia; Female; Animals; Receptors, Somatostatin; Rats; Polyadenylation; RNA-Binding Proteins; Trophoblasts; Cell Proliferation; Epithelial-Mesenchymal Transition; Rats, Sprague-Dawley; Adult; Disease Progression; Cell Movement; Protein Biosynthesis; Placenta
PubMed: 38648900
DOI: 10.1016/j.bbadis.2024.167191 -
PLoS Pathogens Apr 2024Virus discovery by genomics and metagenomics empowered studies of viromes, facilitated characterization of pathogen epidemiology, and redefined our understanding of the...
Virus discovery by genomics and metagenomics empowered studies of viromes, facilitated characterization of pathogen epidemiology, and redefined our understanding of the natural genetic diversity of viruses with profound functional and structural implications. Here we employed a data-driven virus discovery approach that directly queries unprocessed sequencing data in a highly parallelized way and involves a targeted viral genome assembly strategy in a wide range of sequence similarity. By screening more than 269,000 datasets of numerous authors from the Sequence Read Archive and using two metrics that quantitatively assess assembly quality, we discovered 40 nidoviruses from six virus families whose members infect vertebrate hosts. They form 13 and 32 putative viral subfamilies and genera, respectively, and include 11 coronaviruses with bisegmented genomes from fishes and amphibians, a giant 36.1 kilobase coronavirus genome with a duplicated spike glycoprotein (S) gene, 11 tobaniviruses and 17 additional corona-, arteri-, cremega-, nanhypo- and nangoshaviruses. Genome segmentation emerged in a single evolutionary event in the monophyletic lineage encompassing the subfamily Pitovirinae. We recovered the bisegmented genome sequences of two coronaviruses from RNA samples of 69 infected fishes and validated the presence of poly(A) tails at both segments using 3'RACE PCR and subsequent Sanger sequencing. We report a genetic linkage between accessory and structural proteins whose phylogenetic relationships and evolutionary distances are incongruent with the phylogeny of replicase proteins. We rationalize these observations in a model of inter-family S recombination involving at least five ancestral corona- and tobaniviruses of aquatic hosts. In support of this model, we describe an individual fish co-infected with members from the families Coronaviridae and Tobaniviridae. Our results expand the scale of the known extraordinary evolutionary plasticity in nidoviral genome architecture and call for revisiting fundamentals of genome expression, virus particle biology, host range and ecology of vertebrate nidoviruses.
Topics: Animals; Nidovirales; Genome, Viral; Coronavirus; Phylogeny; Vertebrates; Fishes; Evolution, Molecular; Data Mining; Nidovirales Infections
PubMed: 38648214
DOI: 10.1371/journal.ppat.1012163 -
Leukemia Research Jun 2024Acute myeloid leukemia (AML) is a hematopoietic malignancy with a high relapse rate and progressive drug resistance. Alternative polyadenylation (APA) contributes to...
Acute myeloid leukemia (AML) is a hematopoietic malignancy with a high relapse rate and progressive drug resistance. Alternative polyadenylation (APA) contributes to post-transcriptional dysregulation, but little is known about the association between APA and AML. The APA quantitative trait locus (apaQTL) is a powerful method to investigate the relationship between APA and single nucleotide polymorphisms (SNPs). We quantified APA usage in 195 Chinese AML patients and identified 4922 cis-apaQTLs related to 1875 genes, most of which were newly reported. Cis-apaQTLs may modulate the APA selection of 115 genes through poly(A) signals. Colocalization analysis revealed that cis-apaQTLs colocalized with cis-eQTLs may regulate gene expression by affecting miRNA binding sites or RNA secondary structures. We discovered 207 cis-apaQTLs related to AML risk by comparing genotype frequency with the East Asian healthy controls from the 1000 Genomes Project. Genes with cis-apaQTLs were associated with hematological phenotypes and tumor incidence according to the PHARMGKB and MGI databases. Collectively, we profiled an atlas of cis-apaQTLs in Asian AML patients and found their association with APA selection, gene expression, AML risk, and complex traits. Cis-apaQTLs may provide insights into the regulatory mechanisms related to APA in AML occurrence, progression, and prognosis.
Topics: Humans; Quantitative Trait Loci; Leukemia, Myeloid, Acute; Polyadenylation; Polymorphism, Single Nucleotide; Male; Female; Middle Aged; Genetic Predisposition to Disease; Adult; Gene Expression Regulation, Leukemic; Aged; Asian People
PubMed: 38640632
DOI: 10.1016/j.leukres.2024.107499 -
Triblock PolyA-Mediated Protein Biosensor Based on a Size-Matching Proximity Hybridization Analysis.Analytical Chemistry Apr 2024The antibodies in the natural biological world utilize bivalency/multivalency to achieve a higher affinity for antigen capture. However, mimicking this mechanism on the...
The antibodies in the natural biological world utilize bivalency/multivalency to achieve a higher affinity for antigen capture. However, mimicking this mechanism on the electrochemical sensing interface and enhancing biological affinity through precise spatial arrangement of bivalent aptamer probes still pose a challenge. In this study, we have developed a novel self-assembly layer (SAM) incorporating triblock polyA DNA to enable accurate organization of the aptamer probes on the interface, constructing a "lock-and-key-like" proximity hybridization assay (PHA) biosensor. The polyA fragment acts as an anchoring block with a strong affinity for the gold surface. Importantly, it connects the two DNA probes, facilitating one-to-one spatial proximity and enabling a controllable surface arrangement. By precisely adjusting the length of the polyA fragment, we can tailor the distance between the probes to match the molecular dimensions of the target protein. This design effectively enhances the affinity of the aptamers. Notably, our biosensor demonstrates exceptional specificity and sensitivity in detecting PDGF-BB, as confirmed through successful validation using human serum samples. Overall, our biosensor presents a novel and versatile interface for proximity assays, offering a significantly improved surface arrangement and detection performance.
Topics: Biosensing Techniques; Humans; Nucleic Acid Hybridization; Aptamers, Nucleotide; Becaplermin; Poly A; Gold; DNA Probes
PubMed: 38632948
DOI: 10.1021/acs.analchem.4c00210 -
Journal of Virology May 2024Many viruses inhibit general host gene expression to limit innate immune responses and gain preferential access to the cellular translational apparatus for their protein...
Many viruses inhibit general host gene expression to limit innate immune responses and gain preferential access to the cellular translational apparatus for their protein synthesis. This process is known as host shutoff. Influenza A viruses (IAVs) encode two host shutoff proteins: nonstructural protein 1 (NS1) and polymerase acidic X (PA-X). NS1 inhibits host nuclear pre-messenger RNA maturation and export, and PA-X is an endoribonuclease that preferentially cleaves host spliced nuclear and cytoplasmic messenger RNAs. Emerging evidence suggests that in circulating human IAVs NS1 and PA-X co-evolve to ensure optimal magnitude of general host shutoff without compromising viral replication that relies on host cell metabolism. However, the functional interplay between PA-X and NS1 remains unexplored. In this study, we sought to determine whether NS1 function has a direct effect on PA-X activity by analyzing host shutoff in A549 cells infected with wild-type or mutant IAVs with NS1 effector domain deletion. This was done using conventional quantitative reverse transcription polymerase chain reaction techniques and direct RNA sequencing using nanopore technology. Our previous research on the molecular mechanisms of PA-X function identified two prominent features of IAV-infected cells: nuclear accumulation of cytoplasmic poly(A) binding protein (PABPC1) and increase in nuclear poly(A) RNA abundance relative to the cytoplasm. Here we demonstrate that NS1 effector domain function augments PA-X host shutoff and is necessary for nuclear PABPC1 accumulation. By contrast, nuclear poly(A) RNA accumulation is not dependent on either NS1 or PA-X-mediated host shutoff and is accompanied by nuclear retention of viral transcripts. Our study demonstrates for the first time that NS1 and PA-X may functionally interact in mediating host shutoff.IMPORTANCERespiratory viruses including the influenza A virus continue to cause annual epidemics with high morbidity and mortality due to the limited effectiveness of vaccines and antiviral drugs. Among the strategies evolved by viruses to evade immune responses is host shutoff-a general blockade of host messenger RNA and protein synthesis. Disabling influenza A virus host shutoff is being explored in live attenuated vaccine development as an attractive strategy for increasing their effectiveness by boosting antiviral responses. Influenza A virus encodes two proteins that function in host shutoff: the nonstructural protein 1 (NS1) and the polymerase acidic X (PA-X). We and others have characterized some of the NS1 and PA-X mechanisms of action and the additive effects that these viral proteins may have in ensuring the blockade of host gene expression. In this work, we examined whether NS1 and PA-X functionally interact and discovered that NS1 is required for PA-X to function effectively. This work significantly advances our understanding of influenza A virus host shutoff and identifies new potential targets for therapeutic interventions against influenza and further informs the development of improved live attenuated vaccines.
Topics: Humans; A549 Cells; Host-Pathogen Interactions; Influenza A virus; Influenza, Human; RNA, Messenger; Viral Nonstructural Proteins; Virus Replication; Host-Parasite Interactions
PubMed: 38629840
DOI: 10.1128/jvi.01901-23 -
ACS Medicinal Chemistry Letters Apr 2024Chronic hepatitis B (CHB) virus infection afflicts hundreds of millions of people and causes nearly one million deaths annually. The high levels of circulating viral...
Chronic hepatitis B (CHB) virus infection afflicts hundreds of millions of people and causes nearly one million deaths annually. The high levels of circulating viral surface antigen (HBsAg) that characterize CHB may lead to T-cell exhaustion, resulting in an impaired antiviral immune response in the host. Agents that suppress HBsAg could help invigorate immunity toward infected hepatocytes and facilitate a functional cure. A series of dihydropyridoisoquinolizinone (DHQ) inhibitors of human poly(A) polymerases PAPD5/7 were reported to suppress HBsAg in vitro. An example from this class, RG7834, briefly entered the clinic. We set out to identify a potent, orally bioavailable, and safe PAPD5/7 inhibitor as a potential component of a functional cure regimen. Our efforts led to the identification of a dihydropyridophthalazinone (DPP) core with improved pharmacokinetic properties. A conformational restriction strategy and optimization of core substitution led to GS-8873, which was projected to provide deep HBsAg suppression with once-daily dosing.
PubMed: 38628802
DOI: 10.1021/acsmedchemlett.4c00037 -
Hemoglobin Mar 2024Alpha and Beta Thalassemia are autosomal recessive anemias that cause significant morbidity and mortality worldwide, especially in the Middle East and North Africa...
Severe Transfusion-Dependent Thalassemia in Compound Heterozygote Palestinian Siblings with Two α-Globin Gene Defects, Hb Taybe D : C.119_121delCCA Mutation and : C.*94A > G Mutation.
Alpha and Beta Thalassemia are autosomal recessive anemias that cause significant morbidity and mortality worldwide, especially in the Middle East and North Africa (MENA) region where carrier rates reach up to 50%. We report the case of two siblings of Palestinian origin born who presented to our tertiary healthcare center for the management of severe transfusion dependent hemolytic anemia. Before presentation to our center, the siblings were screened for a-thalassemia using the Alpha-globin StripAssay. They were found to carry the α2 polyA-1 [AATAAA > AATAAG] mutation in the heterozygous form, which was insufficient to make a diagnosis. No pathogenic variants were detected on Sanger sequencing of the gene. Full sequencing of the a-gene revealed compound heterozygous variants (:c.119_121delCCA and the previously detected :c.*+94A > G Poly A [A->G]) with trans inheritance. This report highlights the impact of non-deletional mutations on α-globin chain stability. The compound heterozygosity of a rare α-globin chain pathogenic variant with a polyadenylation mutation in the probands leads to clinically severe a-thalassemia. Due to the high carrier status, the identification of rare mutations through routine screening techniques in our populations may be insufficient. Ongoing collaboration among hematologists, medical geneticists, and counselors is crucial for phenotypic-genotypic correlation and assessment of adequate genetic testing schemes.
Topics: Female; Humans; Male; alpha-Globins; alpha-Thalassemia; Arabs; Blood Transfusion; Hemoglobins, Abnormal; Heterozygote; Mutation; Siblings; Child, Preschool; Child
PubMed: 38626925
DOI: 10.1080/03630269.2024.2338850