Development of amplified fragment length polymorphism (AFLP)‐derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot

Aims

The objective of this work was to design an amplified fragment length polymorphism (AFLP)‐derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot (PBRR) in plant material and soil.

Methods and Results

Specific primers for the detection of the pathogen were designed based on an amplified region using AFLPs. The banding patterns by AFLPs showed that isolates from diseased roots were clearly distinguishable from others members of the F. solani species complex. Many bands were specific to F. solani PBRR, one of these fragments was selected and sequenced. Sequence obtained was used to develop specific PCR primers for the identification of pathogen in pure culture and in plant material and soil. Primer pair FS1/FS2 amplified a single DNA product of 175 bp. Other fungal isolates occurring in soil, included F. solani non‐PBRR, were not detected by these specific primers. The assay was effective for the detection of pathogen from diseased root and infected soils.

Conclusions

The designed primers for F. solani causing PBRR can be used in a PCR diagnostic protocol to rapidly and reliably detect and identify this pathogen.

Significance and Impact of the Study

These diagnostic PCR primers will aid the detection of F. solani causing PBRR in diseased root and natural infected soils. The method developed could be a helpful tool for epidemiological studies and to avoid the spread of this serious disease in new areas.     

Index for estimation of muscle force from mechanomyography based on the Lempel–Ziv algorithm

The study of the amplitude of respiratory muscle mechanomyographic (MMG) signals could be useful in clinical practice as an alternative non-invasive technique to assess respiratory muscle strength. The MMG signal is stochastic in nature, and its amplitude is usually estimated by means of the average rectified value (ARV) or the root mean square (RMS) of the signal. Both parameters can be used to estimate MMG activity, as they correlate well with muscle force. These estimations are, however, greatly affected by the presence of structured impulsive noise that overlaps in frequency with the MMG signal. In this paper, we present a method for assessing muscle activity based on the Lempel–Ziv algorithm: the Multistate Lempel–Ziv (MLZ) index. The behaviour of the MLZ index was tested with synthesised signals, with various amplitude distributions and degrees of complexity, and with recorded diaphragm MMG signals. We found that this index, like the ARV and RMS parameters, is positively correlated with changes in amplitude of the diaphragm MMG components, but is less affected by components that have non-random behaviour (like structured impulsive noise). Therefore, the MLZ index could provide more information to assess the MMG–force relationship.     

Development and laboratory evaluation of chemically‐based baited ovitrap for the monitoring of Aedes aegypti

Aedes (Stegomyia) aegypti is considered to be the most important dengue vector worldwide. Studies were conducted to design and evaluate a chemically‐based baited ovitrap for monitoring Ae. aegypti under laboratory conditions. Several known chemical attractants and three types of ovitraps (ovitraps A, B, and C) were evaluated throughout the oviposition bioassays. Oviposition responses of gravid female Ae. aegypti were evaluated to n‐heneicosane, 3‐methylindole (skatole), 4‐methylphenol (p‐cresol), and phenol. Female Ae. aegypti were attracted to all the evaluated compounds. Among them, n‐heneicosane at a concentration of 10 ppm (mg/l), skatole from 50 to 1000 ppm, p‐cresol at 100 ppm, and phenol at 50 ppm showed a significant positive oviposition response. A blend of the four chemical attractants increased the oviposition response; 67% of the eggs were deposited in the treatment compared to the control. Female Ae. aegypti were signi?cantly more attracted to ovitrap A loaded with the four‐component synthetic blend compared to the standard ovitrap in the oviposition bioassays. The compound used in ovitrap A retained its attractant property for up to three days. The chemically‐based baited ovitrap may be considered as an option to be integrated during the monitoring of dengue virus vectors in México.     

Unraveling the multifaceted nature of the nuclear function of mTOR

Positioned at the axis between the cell and its environment, mTOR directs a wide range of cellular activity in response to nutrients, growth factors, and stress. Our understanding of the role of mTOR is evolving beyond the spatial confines of the cytosol, and its role in the nucleus becoming ever more apparent. In this review, we will address various studies that explore the role of nuclear mTOR (nmTOR) in specific cellular programs and how these pathways influence one another. To understand the emerging roles of nuclear mTOR, we discuss data and propose plausible mechanisms to offer novel ideas, hypotheses, and future research directions.     

MicroRNA fate upon targeting with anti-miRNA oligonucleotides as revealed by an improved Northern-blot-based method for miRNA detection

MicroRNAs (miRNAs) are small non-coding RNAs involved in fine-tuning of gene regulation. Antisense oligonucleotides (ONs) are promising tools as anti-miRNA (anti-miR) agents toward therapeutic applications and to uncover miRNA function. Such anti-miR ONs include 2'-O-methyl (OMe), cationic peptide nucleic acids like K-PNA-K3, and locked nucleic acid (LNA)-based anti-miRs such as LNA/DNA or LNA/OMe. Northern blotting is a widely used and robust technique to detect miRNAs. However, miRNA quantification in the presence of anti-miR ONs has proved to be challenging, due to detection artifacts, which has led to poor understanding of miRNA fate upon anti-miR binding. Here we show that anti-miR ON bound to miR-122 can prevent the miRNA from being properly precipitated into the purified RNA fraction using the standard RNA extraction protocol (TRI-Reagent), yielding an RNA extract that does not reflect the real cellular levels of the miRNA. An increase in the numbers of equivalents of isopropanol during the precipitation step leads to full recovery of the targeted miRNA back into the purified RNA extract. Following our improved protocol, we demonstrate by Northern blotting, in conjunction with a PNA decoy strategy and use of high denaturing PAGE, that high-affinity anti-miRs (K-PNA-K3, LNA/DNA, and LNA/OMe) sequester miR-122 without causing miRNA degradation, while miR-122 targeting with a lower-affinity anti-miR (OMe) seems to promote degradation of the miRNA. The technical issues explored in this work will have relevance for other hybridization-based techniques for miRNA quantification in the presence of anti-miR ONs.     

Trivalent PEGylated platform for the conjugation of bioactive compounds

PEGylated multivalent structures are a new class of platform for biological applications due to their biocompatibility properties. Here, we present the synthesis of a trivalent structure 1 based on poly(ethylene glycol) units (PEG) as potential synthetic multifunctional carrier molecule. To evaluate whether this PEGylated platform could be useful for the conjugation of bioactive compounds, a well-known lipopolysaccharide (LPS) inhibitor 2, developed in our laboratory, was selected to be conjugated to 1. The LPS-neutralizing activity of the resulted conjugates and precursors was established using the chromogenic Limulus amebocyte lysate (LAL) assay. The trivalent structure 1 did not show LPS-binding activity, nonconjugate LPS inhibitor 2 showed high LPS-neutralizing activity, and the trivalent conjugate 4 displayed increased LPS-neutralizing activity and a reduced toxicity profile. These results prove the efficacy of this trivalent platform as a multivalent ligand scaffold for biological applications.