Blunt Terminus Research Articles

Exonuclease-III Assisted the Target Recycling Coupling with Hybridization Chain Reaction for Sensitive mecAGene Analysis by Using PGM.

In the field of neonatal infections nursing, methicillin-resistant Staphylococcus aureus (MRSA) is a major bacterial pathogen. Here, we present a portable biosensor for MRSA detection that is both highly sensitive and portable, owing to its implementation on the personal glucose meter (PGM) platform. The H probe was fixed on the magnetic bead for mecA gene analysis. A blunt 3' terminus appeared in the MBs-H probe when the mecA gene was present. Exonuclease-III (Exo-III) recognized the blunt terminus and cleaved it, freeing the mecA gene and so facilitating target recycling. In the meantime, the remaining H probe-initiated hybridization chain reaction (HCR) led to the desired signal amplification. Portable quantitative detection of mecA gene is possible because PGM can read the quantity of invertase tagged on HCR product. After optimizing several experimental parameters, such as the concentration of Exo-III and incubation time, the constructed sensor is extremely sensitive, with a detection limit of 2 CFU/mL. The results from this sensitive PGM-based sensor are in agreement with those obtained from plate counting methods, suggesting that it can be used to accurately assess the MRSA content in artificial clinical samples. In addition, the PGM sensor can significantly cut down on time spent compared to plate counting techniques. The manufactured sensor provides a promising option for accurate identification of pathogenic bacteria.

Colorimetric analysis of C-reactive protein via 'jellyfish' probe-based exonuclease III-assisted multiple-signal recycles.

C-reactive protein (CRP) is a potential biomarker for evaluating inflammatory responses in patients receiving coronary artery bypass graft surgery. Here, the authors depict a sensitive and reliable colorimetric approach for CRP analysis. In this method, an aptamer specifically binds with CRP and an initiator sequence is released from an arch probe to activate signal amplification. The released initiator sequence hybridizes with the toehold section in the 'jellyfish' probe to form a blunt terminus to induce exonuclease III-assisted signal amplification. The method exhibited a low limit of detection of 1.32 ng/ml and high intraday and interday precision for CRP detection. In summary, this colorimetric approach may provide a potential alternative tool for the evaluation of inflammation in patients receiving coronary artery bypass graft and clinical diagnostics of disease.

Laimydorus zehsaziani n. sp., a new member of the genus from northwest of Iran, with additional data on Labronema vulvapapillatum (Meyl, 1954) Loof & Grootaert, 1981 (Dorylaimida: Dorylaimidae).

A new species belonging to the genus Laimydorus, and a known species of the genus Labronema, collected from the rhizosphere of grassland plants in West Azarbaijan province, Iran, are presented. The new species is characterized by its body length (1.56-1.81 mm in females and 1.39-1.57 mm in males), lip region offset by weak constriction and wider than adjacent body (11-14 μm), odontostyle 17-20 μm long, neck length 312-434 μm, vulva longitudinal (V = 42.2-46.5), uterus well-developed and with Z-differentiations, tail long (219-237 μm, c = 6.9-8.3, c´ = 8.2-10) in females, short and convex-conoid with blunt terminus (19-22 μm, c = 68-72.5, c´= 0.77-0.84) in males, spicules 40-45 μm long, and 18-20 contiguous ventromedian supplements with hiatus. Molecular analyses, using Bayesian inference based on D2-D3 expansion segments of the 28S rRNA (LSU), placed the new species in a clade comprising representatives of the genera Nevadanema, Dorylaimus, Labronema, Calcaridorylaimus, Mesodorylaimus, and Prodorylaimus, which all belong to Dorylaimidae. In addition, new data (molecular, morphometrics, images, and geographic distribution) are presented for the Iranian population of Labronema vulvapapillatum.

Neomisticius platypi n. sp. and N. variabilis n. sp. (Tylenchomorpha: Anguinidae) from dead oak trees in Japan

Summary Two new Neomisticius species, typologically and phylogenetically close to each other, are described and illustrated from dead Quercus trees and an ambrosia beetle, Platypus quercivorus. The two species share some stylet morphological characteristics, namely, they both possess a long conus occupying more than half of the total stylet length, a long crustaformeria composed of more than 160 cells (eight rows of more than 20 cells each), and a short and broad female tail with a digitate tip. They are distinguished from each other by N. variabilis n. sp. having a wide, spindle-shaped male bursa with a blunt terminus and N. platypi n. sp. having an oval bursa with a rounded terminus. In addition, the males and females of both species have three large rectal glands and the posterior end of the male testis (distal end of the vas deferens) bears three cells that seemingly function as a valve between the vas deferens and the cloacal tube. These characteristics have not been reported in other tylenchids. Currently, the genus contains only three species: the two new species and N. rhizomorphoides, which has a normal stylet with a short conus, a short crustaformeria, and lacks rectal glands and valve cells in the vas deferens. Therefore, the two new species are readily distinguished from N. rhizomorphoides and, based on the previous definition, may even represent a new genus. However, considering their phylogenetic closeness and biological similarities (e.g., association with ambrosia beetles), the generic definition of Neomisticius was emended to include these new species.

Homogeneous biorecognition reaction-induced assembly of DNA nanostructures for ultrasensitive electrochemical detection of kanamycin antibiotic

By the employment of a homogeneous biorecognition reaction to induce the assembled formation of DNA nanostructures at an electrode, herein we develop a novel biosensing method for the ultrasensitive electrochemical detection of kanamycin (Kana) antibiotic. A DNA complex consisting of Kana-aptamer and a hairpin DNA with an exposed 3′-end was first designed for conducting the homogeneous reaction with Kana in the presence of exonuclease I (Exo I). It resulted in the production of a hairpin DNA with a blunt terminus, which could be used for triggering the assembled formation of a layer of DNA nanostructures with orderly distribution and abundant biotin sites at a gold electrode. Then, high-content methylene blue and horseradish peroxidase (HRP)-functionalized gold nanotags would be captured onto the electrode to realize the electrocatalytic signal transduction. Due to the Exo I and HRP-assisted dual signal amplification, a very low detection limit of 9.1 fg mL−1 was obtained for the Kana assay along with a very wide linear range over five-order of magnitude. Considering the excellent performance of the method, it exhibits a promising prospect for practical applications.

Taxonomy of the Genus Lordellonema Andrássy, 1960 (Nematoda: Dorylaimida: Qudsianematidae) with Description of Three New Species

Three new species of the genus LordellonemaAndrassy, 1960 are described and illustrated. Lordellonema indicumsp. nov. has 0.9–1.1 mm long body; lip region offset by constriction with distinctly protruding labial papillae; odontostyle 13–15 µm long, with aperture occupying about 40–50% of its length; vulva pore-like; tail short, rounded and males absent. Lordellonema paramacrodorumsp. nov. has 1.0–1.2 mm long body; lip region distinctly offset, lips separate, rounded; odontostyle sinuate, 27–29 µm long, with aperture occupying about one-third of its length; vulva transverse; well-developed glandular cells present close to vagina; tail conical with rounded tip. Lordellonema spicularissp. nov. is characterized by its 1.2–1.4 mm long body; lip region angular, offset by constriction; odontostyle 15–16 µm long, aperture occupying 36–44% of its length; vulva transverse; tail convex conoid with blunt terminus; males with non-dorylaimid spicules, 5–7 irregularly spaced ventromedian supplements and lateral guiding pieces absent. Lordellonema porosum is redescribed here after more than five decades from Cabo Verde. L. rapsoides (Heyns et Lagerway, 1965) comb. nov. is redescribed here from Saudi Arabia. A diagnostic compendium of valid species is provided and comment has been made on their biogeography.

Label-free colorimetric detection of divalent mercuric ions (Hg2+) based on T-Hg2+-T structure and exonuclease III dual-recycling and G-quadruplex-hemin DNAzyme amplification

ABSTRACT Detection of divalent mercury attracts great significance in most biological and environmental systems. In this study, a Thymine-divalent mercuric ions-Thymine (T-Hg2+-T) structure based colorimetric assay with Exonuclease III (Exo III) assisted recycling and G-quadruplex-hemin DNAzyme amplification is put forward for Hg2+ monitoring. The Hg2+ can combine with T-T mismatches of oligo-1 to form T-Hg2+-T base pairs, which brings forth a double-helix structure with a blunt terminus. Short mononucleotide fragment (trigger DNA) and Hg2+ could be released as the double-helix structure in oligo-1 is cleaved by Exo III. The released Hg2+ can be captured by another oligo-1, and generate the new cycles. At the same time, signal DNA (oligo-2) can hybridize with trigger DNA to form double-stranded DNA with a blunt terminus that can be cleaved by Exo III to accrue the trigger DNA and guanine (G)-quadruplex fragment. The trigger DNA can hybridize with another signal DNA, which implements the dual-recycling amplification. The G-quadruplex fragment can form the G-quadruplex-hemin DNAzymes, which can catalyze the oxidation of 2, 2ʹ-azino-bis(3-ethylbenzothiazo-line-6-sulfonate) disodium salt (ABTS2-) by H2O2. The color change, served as the signal output, can be measured with an ultraviolet spectrophotometer. The proposed assay allows Hg2+ to be detected in the range of 25 pM to 2000 pM with a detection limit of 7 pM. Moreover, it has been commendably applied to the determination of Hg2+ in environmental samples and biological samples.

A Sensitive Fluorescence Biosensor for Silver Ions (Ag+) Detection Based on C-Ag+-C Structure and Exonuclease III-Assisted Dual-Recycling Amplification.

A C-Ag+-C structure-based fluorescence biosensor with novel combination design of exonuclease III (Exo III) dual-recycling amplification is proposed for the application of silver ions (Ag+) detection. Since oligo-1 involves C-C mismatches, the presence of Ag+ can be captured to form C-Ag+-C base pairs, which results in a double-helix structure with a blunt terminus. The double-helix structure can be cleaved by EXO III to release short mononucleotide fragments (trigger DNA) and Ag+. Released Ag+ can form new bindings with oligo-1, and other trigger DNA can be produced in the digestion cycles. Hybridization with the signal DNA (oligo-2) transforms a trigger DNA into double-stranded DNA with blunt terminus which can be cleaved by Exo III to reproduce the trigger DNA and form guanine- (G-) quadruplex DNA. The trigger DNA returns free to the solution and hybridizes with another signal DNA, which realizes the dual-recycling amplification. The G-quadruplex DNA can be reported by N-methylmesoporphyrin IX (NMM), a specific G-quadruplex DNA fluorochrome. This method allows Ag+ to be determined in the 5 to 1500 pmol/L concentration range, with a 2 pmol/L detection limit, and it has been successfully applied to the detection of Ag+ in real samples.

Paractinolaimus persicus sp. n. (Nematoda: Actinolaimidae) from Iran.

A new species of the genus Paractinolaimus, namely, Paractinolaimus persicus sp. n., is described. It is characterised by body 2.3-2.6 mm long; low, truncate lip region; four massive, acute onchia; stoma walls covered with sparse tiny denticles; odontostyle 25-28 μm long; well developed, didelphic-amphidelphic female reproductive apparatus; vulva a longitudinal oval pore; absence of advulval papillae; female tail 146-224 µm long (c' = 3.3-5.9), elongated filiform, curved ventrad, and with acute terminus; male tail convex conoid with blunt terminus; and 17-18 nearly contiguous ventromedian supplements. This species was recovered from the rhizosphere of wild grass. Line drawings, LM and SEM photographs and measurements are provided.

Ochratoxin A detection platform based on signal amplification by Exonuclease III and fluorescence quenching by gold nanoparticles

This study aimed to establish a detection platform for ochratoxin A (OTA), a common mycotoxin contaminant of various food products. The detection platform is based on signal amplification by Exonuclease III (Exo III) and fluorescence quenching by gold nanoparticles (AuNPs). The aptamer recognizes and binds to OTA, thus leading to the formation of duplex DNA between the complimentary DNA (cDNA) strand and the signal probe. Exo III then digests the duplex DNA from the 3′ blunt terminus of the signal probe to liberate the fluorophore and release cDNA. The released cDNA then hybridizes with other signal probes to initiate a new cleavage reaction. Through this cyclic hybridization–hydrolysis process, an OTA molecule can trigger the cleavage of a large quantity of signal probes. Upon the addition of AuNPs, the fluorophore cannot be adsorbed and quenched, thus notably amplifying fluorescence. The designed aptasensor is highly selective for OTA with a low limit of detection (4.82nM). The feasibility of the detection procedure and the applicability of the aptasensor were validated through the detection of OTA in spiked red wine without interference from the sample matrix. Results indicated that the aptasensor could be used to verify the effectiveness of mycotoxin-control strategies.

Molecular and Morphological Characterization of Xiphinema chambersi Population from Live Oak in Jekyll Island, Georgia, with Comments on Morphometric Variations.

A population of Xiphinema chambersi from the root zone around live oak (Quercus virginiana Mill.) trees on Jekyll Island, GA, is described using both morphological and molecular tools and compared with descriptions of type specimens. Initially, because of a few morphological differences, this nematode was thought to represent an undescribed species. However, on further examination, the morphometrics of the nematodes from live oak tend to agree with most of the morphometrics in the original description and redescription of X. chambersi except for few minor differences in V% relative to body length, slightly shorter stylet length, different c value, and the number of caudal pores. We consider these differences to be part of the normal variation within this species and accordingly image this new population of X. chambersi and redescribe the species. The new population is characterized by having females with a body length of 2.1 to 2.5 mm; lip region slightly rounded and set off from head; total stylet length 170 to 193 µm; vulva at 20.4% to 21.8% of body length; a monodelphic, posterior reproductive system; elongate, conoid tail with a blunt terminus and four pairs of caudal pores, of which two pairs are subdorsal and two subventral. Sequence data from the D2-D3 region of the 28S rRNA molecule subjected to GenBank sequence comparison using BLAST showed that the sequence had 96% and 99% similarity with X. chambersi from Alabama and Florida, respectively. Phylogenetic relationships of X. chambersi with other xiphinematids based on analysis of this DNA fragment are presented. This finding represents a new location of X. chambersi in Georgia on live oak for this species.

The genus Paradorylaimus Andrássy, 1969 (Nematoda: Dorylaimida) with description of three new species from Ecuador

Three new species of Paradorylaimus Andrássy, 1969, found in moss and litter of natural environments of Ecuador aredescribed. P. andinus sp. n. is characterized by 1.65–2.26 mm long female body, lip region set off by a depression, odontostyle26.5–32. 5 μm long and 3.0–4.5 µm wide; pre-rectum 1.1–2.4 anal body diameters long in female and 1.2–2.3 in male; 0–3 pre-vulval and 0–5 postvulval papillae; spicules 60–80 µm long; 13–14 ventral supplements; female tail elongate-filiform, 6–10anal body diameters long; male tail short, 0.7–1 cloacal body diameters long, dorsally convex, with a marked ventral concavityand blunt terminus. P. longicaudatus sp. n. is characterized by 1.78–2.14 mm long female body, lip region slightly set off by adepression, odontostyle 30–32.5 μm long and 2.0–3.5 µm wide; pre-rectum 1.3–2.3 anal body diameters long in female and 2–3.5 in male; paravulval papillae absent; spicules 44.5–62.5 µm long; 11–13 ventral supplements; female tail elongate-filiform,11–16 anal body diameters long; male tail short, 0.8–0.9 cloacal body diameters long, convex conoid, with blunt terminus. P.flagellicaudatus sp. n. is characterized by 1.65–1.73 mm long female body, lip region continuous, odontostyle 19–24 μm longand 2–2.5 µm wide; pre-rectum 1.8–2.6 anal body diameters long in female and 2.4 in male; spicules 45 µm long; 10 ventralsupplements; female tail elongate-filiform, 25–26 anal body diameters long; male tail short, conoid, 0.8 cloacal body diameterslong. The authors support the validity of the genus Paradorylaimus, provide an updated diagnosis and furnish a compendium of the main morphometric parameters of its species and a key to them.

An ultrasensitive fluorescent aptasensor for adenosine detection based on exonuclease III assisted signal amplification

We report here a graphene oxide (GO)-based fluorescent aptasensor for adenosine detection by employing exonuclease III (Exo III) as a signal amplifying element. In the absence of adenosine, the adenosine aptamers hybridized with the complementary DNA (cDNA), and the Exo III could not cleave the single-strand signal probes labeled with carboxylfluorescein (FAM) at its 5′ ends. When the graphene oxide was finally added, it could strongly adsorb the single-strand signal probes and quenched the fluorophore effectively. In the presence of adenosine, the aptamers associated with the targets, which led to the formation of duplex DNAs between the cDNAs and the signal probes. The Exo III thereafter could digest the duplex DNAs from 3′ blunt terminus of signal probes, liberating the fluorophore. Upon adding the GO, the fluorophore could not be adsorbed and quenched. By coupling cyclic enzymatic cleavage, a remarkable fluorescent increase was obtained. Due to the specific recognition ability of the aptamer for the target and the powerful quenching property of GO for signal probe, this proposed approach has a good selectivity and high sensitivity for adenosine. In the optimum conditions described, >100% signal enhancement was achieved and a limit of detection as low as 1nM was obtained, which is lower than those of commonly used fluorescent aptamer sensors. Moreover, the biosensor exhibited an ultrahigh sensitivity and held a versatile platform for clinical diagnostics, molecular biology and drug developments.

Four new and four known species of Tylencholaimoidea (Dorylaimida: Nematoda) from China

Four new and four known species of soil nematodes belonging to the superfamily Tylencholaimoidea Filipjev, 1934 are reported from China. Tylencholaimus orientalis sp. nov. is characterized by having a 0.45–0.61‐mm body; a 5–6‐µm odontostyle; a monoprodelphic genital system with a short post‐uterine sac; and a conoid to hemispheroid tail. Tylencholaimus sinensis sp. nov. is characterized by having a 0.76–0.82‐mm body; a 7–7.5‐µm odontostyle; an amphidelphic genital system; a conoid to hemispheroid tail; and males with 32‐µm spicules and three ventromedians. Proleptonchus sinensis sp. nov. is characterized by having a 1.4–1.7‐mm body; wide amphids; 8–9‐µm odontostyle; a monoprodelphic genital system, the posterior branch with well‐developed uterus and oviduct rudiments; slender and slightly arcuate spicules; and a short conoid tail with thick cuticle at terminus. Dorylaimoides alpinus sp. nov. is characterized by having a 1.4–1.5‐mm body; a lip region offset by a depression; a 12–13‐µm odontostyle; an arcuate odontophore; an amphidelphic genital system with transverse vulva; and a tail that gradually tapers to a blunt terminus.

Cheminformatics methods for novel nanopore analysis of HIV DNA termini.

BackgroundChannel current feature extraction methods, using Hidden Markov Models (HMMs) have been designed for tracking individual-molecule conformational changes. This information is derived from observation of changes in ionic channel current blockade "signal" upon that molecule's interaction with (and occlusion of) a single nanometer-scale channel in a "nanopore detector". In effect, a nanopore detector transduces single molecule events into channel current blockades. HMM analysis tools described are used to help systematically explore DNA dinucleotide flexibility, with particular focus on HIV's highly conserved (and highly flexible/reactive) viral DNA termini. One of the most critical stages in HIV's attack is the binding between viral DNA and the retroviral integrase, which is influenced by the dynamic-coupling induced high flexibility of a CA/TG dinucleotide positioned precisely two base-pairs from the blunt terminus of the duplex viral DNA. This suggests the study of a family of such CA/TG dinucleotide molecules via nanopore measurement and cheminformatics analysis.ResultsHMMs are used for level identification on the current blockades, HMM/EM with boosted variance emissions are used for level projection pre-processing, and time-domain FSAs are used to parse the level-projected waveform for kinetic information. The observed state kinetics of the DNA hairpins containing the CA/TG dinucleotide provides clear evidence for HIV's selection of a peculiarly flexible/interactive DNA terminus.

Substrate Specificity for 4-Thiouridine Modification in Escherichia coli

The biosynthesis of 4-thiouridine (s4U) in Escherichia coli tRNA requires the action of both the thiamin pathway enzyme ThiI and the cysteine desulfurase IscS. IscS catalyzes sulfur transfer from l-cysteine to ThiI, which utilizes Mg-ATP to activate uridine 8 in tRNA and transfers sulfur to give s4U. In this work, we show through deletion analysis of unmodified E. coli tRNA(Phe) that the minimum substrate for s4U modification is a mini-helix comprising the stacked acceptor and T stems containing an internal bulged region. The size of the bulged loop must be at least 4 nucleotides and contain the target uridine as the first nucleotide. Replacement of the T loop sequence with a tetraloop in the deletion substrate increases activity and shows that the TpsiC primary sequence is not a recognition element. An unmodified tRNA(Phe) transcript in which the 3'-terminal ACCA sequence is removed to give a blunt terminus has <0.1% activity, although the addition of a single overhanging base essentially restores activity. In addition, reducing the distance of the 3' terminus relative to U8 by as little as 1 bp severely impairs activity. By dissecting a minimal RNA substrate in the T loop region, a two-piece system consisting of a substrate RNA and a "guide" RNA is efficiently modified. Our results indicate that outside of the modified U8, there is no primary sequence requirement for substrate recognition. However, the secondary and tertiary structure restrictions appear sufficient to explain why s4U modification is limited in the cell to tRNA.

Two New Species of the Genus Dorylaimoides Thorne &amp; Swanger, 1936 (Nematoda: Dorylaimida) From Spain

Two new species belonging to the genus Dorylaimoides Thorne & Swanger, 1936 are described from natural areas in southeastern Spain, D. baeticus sp. n. is characterized by having lip region offset from the adjacent body, odontostyle shorter than head diameter, pharyngeal bulb marked off by a constriction, anterior genital branch reduced to a simple sac 1.7-3.5 times the corresponding body width long, tail conical with blunt terminus and five ventromedian supplements regularly spaced. D. cylindricaudatus sp. n. is distinguished by medium size (1.18-1.38 mm long), lips amalgamated with low inner part, pharyngeal bulb constricted, female genital system pseudo-opisthodelphic, tail cylindrical elongated 3.8-5 anal body widths long,and 5-6 regularly spaced ventromedian supplements.

Mehdilaimus a Urangabadensis N.Gen., N.Sp. (Axonolaimidae: Cylindrolaiminae) From Marathwada, India

Mehdilaimus aurangabadensis n. gen., n. sp. was collected from soil around the roots of sugar-beet. It differs from Cylindrolaimus de Man, 1880 in lacking head setae; in having the oesophagus one and a half times longer than the stoma, the former being almost of equal width throughout its length; in having a prodelphic reflexed gonad, and in having a conoid tail with blunt rounded terminus and without terminal gland and duct. From Aulolaimus de Man, 1880 it differs in lacking a markedly narrowed head end, longitudinal striations on cuticle, oval amphids, amphidelphic gonad and long filiform tail.

4thiouridine-specific spin-labeling of [formula omitted] transfer RNA

The biosynthesis of 4-thiouridine (s4U) in Escherichia coli tRNA requires the action of both the thiamin pathway enzyme ThiI and the cysteine desulfurase IscS. IscS catalyzes sulfur transfer from l-cysteine to ThiI, which utilizes Mg-ATP to activate uridine 8 in tRNA and transfers sulfur to give s4U. In this work, we show through deletion analysis of unmodified E. coli tRNAPhe that the minimum substrate for s4U modification is a mini-helix comprising the stacked acceptor and T stems containing an internal bulged region. The size of the bulged loop must be at least 4 nucleotides and contain the target uridine as the first nucleotide. Replacement of the T loop sequence with a tetraloop in the deletion substrate increases activity and shows that the TψC primary sequence is not a recognition element. An unmodified tRNAPhe transcript in which the 3′-terminal ACCA sequence is removed to give a blunt terminus has <0.1% activity, although the addition of a single overhanging base essentially restores activity. In addition, reducing the distance of the 3′ terminus relative to U8 by as little as 1 bp severely impairs activity. By dissecting a minimal RNA substrate in the T loop region, a two-piece system consisting of a substrate RNA and a “guide” RNA is efficiently modified. Our results indicate that outside of the modified U8, there is no primary sequence requirement for substrate recognition. However, the secondary and tertiary structure restrictions appear sufficient to explain why s4U modification is limited in the cell to tRNA.