Strategies for signal amplification in nucleic acid detection

Many aspects of molecular genetics necessitate the detection of nucleic acid sequences. Current approaches involving target amplification (in situ PCR, Primed in situ Labeling, Self-Sustained Sequence Replication, Strand Displacement Amplification), probe amplification (Ligase Chain Reaction, Padlock Probes, Rolling Circle Amplification) and signal amplification (Tyramide Signal Amplification, Branched DNA Amplification) are summarized in the present review, together with their advantages and limitations.     

Sample preparation and high-resolution separation of mycotoxins possessing carboxyl groups

The chromatographic analysis of carboxyl-containing mycotoxins, such as fumonisin B₁, ochratoxin A, and citrinin, presents a continual challenge. Toxins must first be extracted from foods or tissues and then cleaned up before chromatographic separation and detection. Liquid–liquid extraction efficiencies for some carboxylic mycotoxins are marginal for spiked samples and uncertain for incurred residues. Immunoaffinity columns may be useful for concentrating mycotoxins from samples before chromatography. In almost every case, more than one analytical method must be used to confirm the identification of the mycotoxin. The fumonisins are especially troublesome to analyze because they are relatively insoluble in organic solvents, they are not separated easily by gas chromatography, and they do not respond to the usual absorbance or fluorescence detectors used in liquid chromatography. Fluorescence derivatization and electrospray liquid chromatography–mass spectrometry have now made it possible to detect trace levels of mycotoxins. The purity of mycotoxin standards for toxicological studies can be determined by liquid chromatography with either an evaporative light scattering detector or electrospray mass spectrometer. New developments in capillary electrophoresis, nonporous microsphere liquid chromatography, and detection methods for low-volatility compounds show promise for improving the analysis of mycotoxins in the future.     

Angiotensinogen promoter and angiotensinogen II receptor type 1 gene polymorphisms and incidence of ischemic stroke and neurologic phenotype in Fabry disease

Abstract

Context: Stroke and/or white matter lesions (WMLs) are significant in Fabry disease. Polymorphisms of angiotensinogen (AGT), AGT Promoter and angiotensinogen II receptor type 1 (AGTR1) are correlated with WMLs.

Objectives: We compared AGT. AGT Promoter and AGTR1 genotypes to stroke incidence, Fabry-specific [Mainz Severity Score Index (MSSI)] severity score, and neurologic sub-score (n-MSSI).

Methods: Sixty-three Fabry patients and 49 matched controls plus historic controls were genotyped. Institutional Review Board approval was received.

Results. C and/or CC alleles of AGT Promoter and AGTR1 were significantly correlated with stroke and n-MSSI.

Discussion/conclusion: Findings are suggestive of role of AGT Promoter and AGTR1 genotypes in Fabry phenotypes.     

Resistance training-induced muscle hypertrophy is mediated by TGF-β1-Smad signaling pathway in male Wistar rats

The TGF-β1-Smad pathway is a well-known negative regulator of muscle growth; however, its potential role in resistance training-induced muscle hypertrophy is not clear. The present study proposed to determine whether and how this pathway may be involved in resistance training-induced muscle hypertrophy. Skeletal muscle samples were collected from the control, trained (RT), control + SB431542 (CI_TGF), and trained + SB431542 (RTI_TGF) animals following 3, 5, and 8 weeks of resistance training. Inhibition of the TGF-β1-Smad pathway by SB431542 augmented muscle satellite cells activation, upregulated Akt/mTOR/S6K1 pathway, and attenuated FOXO1 and FOXO3a expression in the CI_TGF group (all p < .01), thereby causing significant muscle hypertrophy in animals from the CI_TGF. Resistance training significantly decreased muscle TGF-β1 expression and Smad3 (P-Smad3^S423/425) phosphorylation at COOH-terminal residues, augmented Smad2 (P-Smad2-L^S245/250/255) and Smad3 (P-Smad3-L^Ser208) phosphorylation levels at linker sites (all p < .01), and led to a muscle hypertrophy which was unaffected by SB431542, suggesting that the TGF-β1-Smad signaling pathway is involved in resistance training-induced muscle hypertrophy. The effects of inhibiting the TGF-β1-Smad signaling pathway were not additive to the resistance training effects on FOXO1 and FOXO3a expression, muscle satellite cells activation, and the Akt/mTOR/S6K1 pathway. Resistance training effect of satellite cell differentiation was independent of the TGF-β1-Smad signaling pathway. These results suggested that the effect of the TGF-β1-Smad signaling pathway on resistance training-induced muscle hypertrophy can be attributed mainly to its diminished inhibitory effects on satellite cell activation and protein synthesis. Suppressed P-Smad3^S423/425 and enhanced P-Smad2-L^S245/250/255 and P-Smad3-L^Ser208 are the molecular mechanisms that link the TGF-β1-Smad signaling pathway to resistance training-induced muscle hypertrophy.     

The systematic approach to describing conformational rearrangements in G-quadruplexes

Conformational changes in DNA G-quadruplex (GQ)-forming regions affect genome function and, thus, compose an interesting research topic. Computer modelling may yield insight into quadruplex folding and rearrangement, particularly molecular dynamics simulations. Here, we show that specific parameters, which are distinct from those commonly used in DNA conformational analyses, must be introduced for adequate interpretation and, most importantly, convenient visual representation of the quadruplex modelling results. We report a set of parameters that comprehensively and systematically describe GQ geometry in dynamics. The parameters include those related to quartet planarity, quadruplex twist, and quartet stacking; they are used to quantitatively characterise various types of quadruplexes and rearrangements, such as quartet distortion/disruption or deviation/bulging of a single nucleotide from the quartet plane. Our approach to describing conformational changes in quadruplexes using the new parameters is exemplified by telomeric quadruplex rearrangement, and the benefits of applying this approach to analyse other structures are discussed.     

LncRNA NEAT1 promotes docetaxel resistance in prostate cancer by regulating ACSL4 via sponging miR-34a-5p and miR-204-5p

Docetaxel resistance remains one of the main problems in clinical treatment of metastatic prostate cancer (PCa). Previous studies identified differently expressed lncRNAs in docetaxel-resistant PCa cell lines, while the potential mechanisms were still unknown. In the present study, we found NEAT1 was expressed at high levels in docetaxel-resistant PCa clinical samples and related cell lines. When knockdown NEAT1, cell proliferation and invasion in docetaxel-resistant PCa cells in vitro and in vivo were downregulated. Our further researches explained that NEAT1 exerts oncogenic function in PCa by competitively ‘sponging’ both miR-34a-5p and miR-204-5p. Inhibition of miR-34a-5p or miR-204-5p expression mimics the docetaxel-resistant activity of NEAT1, whereas ectopic expression of miR-34a-5p or miR-204-5p attenuates the anti-drug function of NEAT1 in PCa cells. Besides, we also found ACSL4 is a target of both miR-34a-5p and miR-204-5p, and ACSL4 was also inhibited by miR-34a-5p and miR-204-5p. Moreover, suppression of miR-34a-5p or/and miR-204-5p greatly restrained the expression of ACSL4 upon NEAT1 overexpression. Joint expression of miR-34a-5p and miR-204a-5p synergistically decreased the expression of ASCL4, indicating miR-34a-5p and miR-204a-5p collaboratively inhibit the expression of ACSL4. Innovatively, we concluded that NEAT1 contributes to the docetaxel resistance by increasing ACSL4 via sponging miR-34a-5p and miR-204-5p in PCa cells.     

Identification, sequencing and mutagenesis of the gene for a d-carbamoylase from Agrobacterium radiobacter

Abstract A clone positive for d-carbamoylase activity (2.7 kb Hin dIII- Bam H1 DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli . This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The d-carbamoylase gene. named cauA , was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21 (DE3). After induction with isopropyl-thio-β-d-galactopyranoside, the synthesis of d-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced allowed us to establish that a Pro¹⁴→Leu¹⁴ exchange leads to an inactive enzyme species, while a Cys²⁷⁹→Ser²⁷⁹ exchange did not impair the functional properties of the enxyme.     

Conjugation of phosphonoacetic acid to nucleobase promotes a mechanism-based inhibition

Small molecule inhibitors have a powerful blocking action on viral polymerases. The bioavailability of the inhibitor, nevertheless, often raise a significant selectivity constraint and may substantially limit the efficacy of therapy. Phosphonoacetic acid has long been known to possess a restricted potential to block DNA biosynthesis. In order to achieve a better affinity, this compound has been linked with natural nucleotide at different positions. The structural context of the resulted conjugates has been found to be crucial for the acquisition by DNA polymerases. We show that nucleobase-conjugated phosphonoacetic acid is being accepted, but this alters the processivity of DNA polymerases. The data presented here not only provide a mechanistic rationale for a switch in the mode of DNA synthesis, but also highlight the nucleobase-targeted nucleotide functionalization as a route for enhancing the specificity of small molecule inhibitors.     

Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus

Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.