Integrative taxonomy and phylogenetic relationships between representatives of genera within Merliniinae (Nematoda: Merliniidae), with new data on fourteen known and one unknown species.

The application of integrative taxonomic approaches is useful to species delineation based on a combination of distinct types of characters, here morphological features and ribosomal DNA sequences. In this study, we surveyed ectoparasitic nematodes of the subfamily Merliniinae in cultivated and natural environments in Iran. Results of morphological and morphometrical studies, light and scanning electron microscopic observations, and molecular analyses allowed us the identification of fourteen known and one unknown species including representatives of the genera Amplimerlinius (five species), Geocenamus (one species), Merlinius (three species), Nagelus (two species), Paramerlinius (one species), Scutylenchus (two species), and Telomerlinius (one species). The unknown species, Scutylenchus sp., characterized by having 35-50 incisures at mid-body; lateral field with 6 longitudinal incisures; lip region slightly offset by a constriction, flattened at front end; bearing 5-7 annuli; cephalic framework not refractive; stylet robust, 18.3-27 µm long; post anal intestinal sac absent; tail elongate conical, dorsally convex, with 24 (19-28) annuli in ventral side, ending to a smooth terminus and males common; spicules 24.5-31 µm long. The phylogenetic analyses were carried out using molecular data from nuclear ribosomal DNA (rDNA) genes viz. D2-D3 expansion segments of the large ribosomal subunit (28S rRNA), partial small ribosomal subunit (18S rRNA), and internal transcribed spacer (ITS). The molecular variability of D2-D3 expansion segments of the 28S rRNA and partial 18S rRNA was low in this family in comparison to the ITS region, which could be a more helpful molecular marker in species and genus identification.

A higher protein intake at breakfast and lunch is associated with a higher total daily protein intake in older adults: a post-hoc cross-sectional analysis of four randomised controlled trials.

BACKGROUND: A protein intake of 30-40 g per meal is suggested to maximally stimulate muscle protein synthesis in older adults and could therefore contribute to the prevention of sarcopenia. Protein intake at breakfast and lunch is often low and offers a great opportunity to improve daily protein intake. Protein, however, is known for its satiating effects. Therefore, we explored the association between the amount of protein intake at breakfast and lunch and total daily protein intake in older adults. METHODS: Protein intake was assessed by a 3-day food record in 498 community dwelling older adults (≥55 years) participating different lifestyle interventions. Linear mixed model analysis was used to examine the association between protein intake at breakfast or lunch and total daily protein intake, adjusted for sex, age, body mass index, smoking status, study and total energy intake. RESULTS: After adjustment for potential confounders, a 10 g higher protein intake at breakfast was associated with a 3.2 g higher total daily protein intake (P = 0.008) for males and a 4.9 g (P < 0.001) higher total daily protein intake for females. A 10 g higher protein intake at lunch was associated with a 3.7 g higher total daily protein intake (P < 0.001) for males, and a 5.8 g higher total daily protein intake (P < 0.001) for females. CONCLUSIONS: A higher protein intake at breakfast and lunch is associated with a higher total daily protein intake in community dwelling older adults. Stimulating a higher protein intake at breakfast and lunch might represent a promising nutritional strategy to optimise the amount of protein per meal without compromising total daily protein intake.

Reduced graphene oxide, but not carbon nanotubes, slows murine melanoma after thermal ablation using LED light in B16F10 lineage cells.

Photodynamic therapy is a minimally invasive health technology used to treat cancer and other non-malignant diseases, as well as inactivation of viruses, bacteria and fungi. In this work, we sought to combine the phototherapy technique using low intensity LED (660 nm) to induce ablation in melanoma tumor in mice treated with nanoparticles. In vitro and in vivo studies were conducted, and our results demonstrated that multi-walled carbon nanotubes (MWCNTs) do not destroy tumor cells in vivo, but stimulate the inflammatory process and angiogenesis. Reduced graphene oxide (rGO), has been shown to play a protective role associated with the LED ablation, inducing necrosis, stimulation of immune response by lymphoproliferation, and decreased tumor mass in vivo. We consider that LED alone can be very effective in controlling the growth of melanoma tumors and its association with rGO is potentiated.

First Report of Plant-Parasitic Nematode Meloidoderita salina in the Netherlands.

After the description of the root-parasitic nematode Meloidoderita salina from a tidal salt marsh in France (1), an additional sampling was carried out to search for the presence of this unusual nematode in a tidal salt marsh area close to Sint-Annaland, Zeeland Province, the Netherlands. In August and October 2012, a total of 25 soil and root samples were collected from the halophytic plants Atriplex portulacoides L. (so far the only known host for this nematode species), A. littoralis L., A. prostrata Boucher ex DC., Limonium vulgare Mill., Salicornia europaea L., Aster tripolium L., and Plantago maritima L. All these halophytes grow in a cohesive muddy soil type within the salt marsh, except A. littoralis and A. prostrata, which grow in the litter tidal zones on the edges of this area. Nematodes from roots and soil were extracted by centrifugal flotation (2) and Oostenbrink's cotton-wool filter methods (4), respectively. Additionally, roots were used for direct observation of females and young cystoids with a dissecting microscope. Finally, all stages were compared morphologically with available type material (1). Root and soil samples demonstrated that only nematodes isolated from A. portulacoides, A. littoralis, and A. prostrata contained all life stages of the genus Meloidoderita, while on the roots of L. vulgare, S. europaea, A. tripolium, and P. maritima, no Meloidoderita was observed. The soil samples included males, cystoids, and second-stage juveniles (J2) in low densities (<20 nematodes/100 ml), while swollen females and young cystoids were observed on root samples. All stages (n = 10 per life stage) fit morphologically with the recently described M. salina. Females were swollen with an oval to pear shaped body with a small posterior protuberance, irregular and twisted neck, oval and backwardly sloping stylet knobs, a prominent secretory-excretory (S-E) pore with cuticular lobes, and a swollen uterus with a thick hyaline wall. Males were without stylet, strongly sclerotized S-E duct, and tail tapering to rounded terminus ending in one or two ventrally terminal mucron. J2s had a well-developed stylet and rounded knobs set off from shaft and conical tail slightly curved ventrally and tapering to a finely pointed terminus with a finger-like projection. Cystoids showing the unique sub-cuticular hexagonal beaded pattern (1). J2s were also used for molecular analysis. DNA was extracted by incubating individual J2s in a lysis buffer as described in (3). Two primer combinations were used to amplify the small subunit ribosomal DNA (SSU rDNA) from a 100-times-diluted crude lysate (two overlapping fragments, [3]). The resulting (nearly) full-length SSU-rDNA sequences (GenBank KF751617 and KF751618) showed >99% identity with M. salina sequences from nematodes collected in the aforementioned tidal salt marsh in France (FJ969126 and FJ969127). To our knowledge, this is the first report of M. salina in the Netherlands. Moreover, this is the first record of M. salina parasitizing A. littoralis and A. prostrata. Although these Atriplex species are used for human consumption, the effect of M. salina on the host is unknown so far. References: (1) S. Ashrafi et al. Zookeys 249:1, 2012. (2) W. A. Coolen. Pages 317-329 in: Root-knot nematodes (Meloidogyne species). Systematics, biology and control. Academic Press, New York, 1979. (3) Holterman et al. Mol. Biol. Evol. 23:1792, 2006. (4) M. Oostenbrink. Pages 85-102 in: Nematology. University of North Carolina Press, Chapel Hill, 1960.

Using a Kirschner Wire as a Stylet for the Management of a Difficult Neonatal Airway: A Case Report.

We report the successful management of a difficult airway in an extremely low birth weight neonate (700 g) using a Kirschner wire as a substitute for an endotracheal tube stylet. Several intubation attempts were unsuccessful because of the difficulty in guiding a very small and malleable tube under the epiglottis. This study highlights that every maternity hospital should be prepared to manage airways in unexpected extremely low birth weight neonates. Appropriate size equipment and protocols should be readily available.

Up-regulation of T lymphocyte and antibody production by inflammatory cytokines released by macrophage exposure to multi-walled carbon nanotubes.

Our data demonstrate that multi-walled carbon nanotubes (MWCNTs) are internalized by macrophages, subsequently activating them to produce interleukin (IL)-12 (IL-12). This cytokine induced the proliferative response of T lymphocytes to a nonspecific mitogen and to ovalbumin (OVA). This increase in the proliferative response was accompanied by an increase in the expression of pro-inflammatory cytokines, such as interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα) and IL-6, in mice inoculated with MWCNTs, whether or not they had been immunized with OVA. A decrease in the expression of transforming growth factor-beta (TGFß) was observed in the mice treated with MWCNTs, whereas the suppression of the expression of both TGFß and IL-10 was observed in mice that had been both treated and immunized. The activation of the T lymphocyte response by the pro-inflammatory cytokines leads to an increase in antibody production to OVA, suggesting the important immunostimulatory effect of carbon nanotubes.

Effects of multi-walled carbon nanotubes (MWCNT) under Neisseria meningitidis transformation process.

BACKGROUND: This study aimed at verifying the action of multi-walled carbon nanotubes (MWCNT) under the naturally transformable Neisseria meningitidis against two different DNA obtained from isogenic mutants of this microorganism, an important pathogen implicated in the genetic horizontal transfer of DNA, causing the escape of the principal vaccination measured worldwide by the capsular switching process. MATERIALS AND METHODS: The bacterium receptor strain C2135 was cultivated and had its mutant DNA donor M2 and M6, which received a receptor strain and MWCNT at three different concentrations. The inhibition effect of DNAse on the DNA in contact with nanoparticles was evaluated. RESULTS: The results indicated an in increase in the transformation capacity of N. meninigtidis in different concentrations of MWCNT when compared with negative control without nanotubes. A final analysis of the interaction between DNA and MWCNT was carried out using Raman Spectroscopy. CONCLUSION: These increases in the transformation capacity mediated by MWCNT, in meningococci, indicate the interaction of these particles with the virulence acquisition of these bacteria, as well as with the increase in the vaccination escape process.

AC-conductance and capacitance measurements for ethanol vapor detection using carbon nanotube-polyvinyl alcohol composite based devices.

We report the preparation of inexpensive ethanol sensor devices using multiwalled carbon nanotube-polyvinyl alcohol composite films deposited onto interdigitated electrodes patterned on phenolite substrates. We investigate the frequency dependent response of the device conductance and capacitance showing that higher sensitivity is obtained at higher frequency if the conductance is used as sensing parameter. In the case of capacitance measurements, higher sensitivity is obtained at low frequency. Ethanol detection at a concentration of 300 ppm in air is demonstrated. More than 80% of the sensor conductance and capacitance variation response occurs in less than 20 s.

PAH source differentiation between historical MGP and significant urban influences for sediments in San Francisco Bay.

A forensic source evaluation of polycyclic aromatic hydrocarbons (PAHs) in nearshore sediments in San Francisco Bay examined total PAH greater than ambient concentrations in sediments, and potential pyrogenic source relationships with respect to PAH compounds typically associated with point and nonpoint pyrogenic source types, including PAHs potentially associated with historical manufactured gas plant (MGP) operations. Diagnostic source ratio analysis was employed for determination of potential PAH source relationships. A two-model approach indicated distinct potential source signatures, as identified from the distributions of higher PAH concentrations in some sediments. Source characterization was aided by Polytopic Vector Analysis (PVA) and data visualization with t-Distributed Stochastic Neighbor Embedding (t-SNE). Two signatures exhibited pyrogenic character likely consistent with historical MGP sources, and one signature was related to creosote. A distinct and significant source of PAHs to the investigation area sediment consisted of ubiquitous nonpoint and potential unidentified point sources is termed "urban influence".

Amino acid sequence and distribution of mRNA encoding a major skeletal muscle laminin binding protein: an extracellular matrix-associated protein with an unusual COOH-terminal polyaspartate domain.

Two cDNAs encoding an abundant chicken muscle extracellular matrix (ECM)-associated laminin-binding protein (LBP) have been isolated and sequenced. The predicted primary amino acid sequence includes a probable signal peptide and a site for N-linked glycosylation, but lacks a hydrophobic segment long enough to span the membrane. The COOH terminus consists of an unusual repeat of 33 consecutive aspartate residues. Comparison with other sequences indicates that this protein is different from previously described LBPs and ECM receptors. RNA blot analysis of LBP gene expression showed that LBP mRNA was abundant in skeletal and heart muscle, but barely detectable in other tissues. Blots of chicken genomic DNA suggest that a single gene encodes this LBP. The amino acid sequence and mRNA distribution are consistent with the biochemical characterization described by Hall and co-workers (Hall, D. E., K. A. Frazer, B. C. Hahn, and L. F. Reichardt. 1988. J. Cell Biol. 107:687-697). These analyses indicate that LBP is an abundant ECM-associated muscle protein with an unusually high negative charge that interacts with both membranes and laminin, and has properties of a peripheral, not integral membrane protein. Taken together, our studies show that muscle LBP is a secreted, peripheral membrane protein with an unusual polyaspartate domain. Its laminin and membrane binding properties suggest that it may help mediate muscle cell interactions with the extracellular matrix. We propose the name "aspartactin" for this LBP.

Small subunit rDNA-based phylogeny of the Tylenchida sheds light on relationships among some high-impact plant-parasitic nematodes and the evolution of plant feeding.

Cyst (Heteroderidae), root knot (Meloidogyne spp.), and lesion (Pratylenchus spp.) nematodes all belong to a single nematode order, Tylenchida. However, the relationships between and within these economically highly relevant groups, and their relatedness to other parasitic Tylenchida is unclear. We constructed a phylogeny of 116 Tylenchida taxa based on full length small subunit ribosomal DNA (small subunit [SSU] rDNA) sequences. Ancestral state reconstruction points at a gradual development of simple to more complex forms of plant parasitism. Good resolution was observed in distal clades that include cyst, root knot, and lesion nematodes, and monophyly of most families was confirmed. Our data suggest that root knot nematodes have evolved from an ancestral member of the genus Pratylenchus, but it remains unclear which species is closest to this branching point. Contrary to the notoriously polyphagous distal representatives, basal members of the genus Meloidogyne (and probably, their common ancestor) have narrow host ranges. Our analysis also shows that mitotic parthenogeny has arisen at least two times independently among root knot nematodes. In many cases resolution till species was observed, suggesting that SSU rDNA sequences have a potential for DNA barcode-based species identification with, due to the overall conserved nature of this gene, limited intra-species variation.

Structure and developmental expression of the nerve growth factor receptor in the chicken central nervous system.

Chicken nerve growth factor (NGF) receptor cDNAs have been isolated and sequenced in an effort to identify functionally important receptor domains and as an initial step in determining the functions of the NGF receptor in early embryogenesis. Comparisons of the primary amino acid sequences of the avian and mammalian NGF receptors have identified several discrete domains that differ in their degree of conservation. The highly conserved regions include an extracellular domain, likely to be involved in ligand binding, in which the positions of 24 cysteine residues and virtually all negatively charged residues are conserved; a transmembrane region, including flanking stretches of extracellular and cytoplasmic amino acids, which has properties suggesting it interacts with other proteins; and a cytoplasmic PEST sequence, which may regulate receptor turnover. Transient expression of NGF receptor mRNA has been seen in many regions of the developing CNS. Experiments suggest that both NGF and its receptor help regulate development of the retina.

GenEST, a powerful bidirectional link between cDNA sequence data and gene expression profiles generated by cDNA-AFLP.

The release of vast quantities of DNA sequence data by large-scale genome and expressed sequence tag (EST) projects underlines the necessity for the development of efficient and inexpensive ways to link sequence databases with temporal and spatial expression profiles. Here we demonstrate the power of linking cDNA sequence data (including EST sequences) with transcript profiles revealed by cDNA-AFLP, a highly reproducible differential display method based on restriction enzyme digests and selective amplification under high stringency conditions. We have developed a computer program (GenEST) that predicts the sizes of virtual transcript-derived fragments (TDFs) of in silico-digested cDNA sequences retrieved from databases. The vast majority of the resulting virtual TDFs could be traced back among the thousands of TDFs displayed on cDNA-AFLP gels. Sequencing of the corresponding bands excised from cDNA-AFLP gels revealed no inconsistencies. As a consequence, cDNA sequence databases can be screened very efficiently to identify genes with relevant expression profiles. The other way round, it is possible to switch from cDNA-AFLP gels to sequences in the databases. Using the restriction enzyme recognition sites, the primer extensions and the estimated TDF size as identifiers, the DNA sequence(s) corresponding to a TDF with an interesting expression pattern can be identified. In this paper we show examples in both directions by analyzing the plant parasitic nematode Globodera rostochiensis. Various novel pathogenicity factors were identified by combining ESTs from the infective stage juveniles with expression profiles of approximately 4000 genes in five developmental stages produced by cDNA-AFLP.

Establishment of optimal conditions for long-term culture of erythrocytic stages of Theileria uilenbergi.

OBJECTIVE: To establish optimal conditions for long-term culture of the erythrocytic stage of Theileria uilenbergi. SAMPLE POPULATION: Red blood cells from 3 splenectomized sheep experimentally infected with a blood stabilate of T uilenbergi. PROCEDURES: Cultures of T uilenbergi were initiated by use of blood from experimentally infected sheep collected when parasites were detected in Giemsa-stained thin blood smears. Different culture conditions were tested to optimize in vitro growth of the organisms. Subcultures were performed at a ratio of 1:2, 1:4, and 1:8 when the percentage of parasitized erythrocytes (PPE) was at least 1% or when the initial PPE was doubled. RESULTS: The optimal culture medium was HL-1 medium (a complete chemically defined medium) supplemented with 20% sheep serum and 0.75% chemically defined lipids. Optimal culture conditions included incubation in a humidified 2% O(2), 5% CO(2), and 93% N(2) atmosphere at 37 degrees C. Cultures of the merozoite stage of the parasite were continuously propagated in vitro for > 1 year. The PPE reached values of up to 3%. CONCLUSIONS AND CLINICAL RELEVANCE: Optimization of culture conditions to reach a high PPE seems worthwhile. The continuous propagation of T uilenbergi in culture allows the production of parasite material without infecting animals and provides a continuous laboratory source of parasites for further studies.

Therapeutic angiogenesis induced by human umbilical cord tissue-derived mesenchymal stromal cells in a murine model of hindlimb ischemia.

BACKGROUND: Mesenchymal stem cells derived from human umbilical cord tissue, termed UCX®, have the potential to promote a full range of events leading to tissue regeneration and homeostasis. The main goal of this work was to investigate UCX® action in experimentally induced hindlimb ischemia (HLI). METHODS: UCX®, obtained by using a proprietary technology developed by ECBio (Amadora, Portugal), were delivered via intramuscular injection to C57BL/6 females after unilateral HLI induction. Perfusion recovery, capillary and collateral density increase were evaluated by laser doppler, CD31 immunohistochemistry and diaphonisation, respectively. The activation state of endothelial cells (ECs) was analysed after EC isolation by laser capture microdissection microscopy followed by RNA extraction, cDNA synthesis and quantitative RT-PCR analysis. The UCX®-conditioned medium was analysed on Gallios flow cytometer. The capacity of UCX® in promoting tubulogenesis and EC migration was assessed by matrigel tubule formation and wound-healing assay, respectively. RESULTS: We demonstrated that UCX® enhance angiogenesis in vitro via a paracrine effect. Importantly, after HLI induction, UCX® improve blood perfusion by stimulating angiogenesis and arteriogenesis. This is achieved through a new mechanism in which durable and simultaneous upregulation of transforming growth factor ß2, angiopoietin 2, fibroblast growth factor 2, and hepatocyte growth factor, in endothelial cells is induced by UCX®. CONCLUSIONS: In conclusion, our data demonstrate that UCX® improve the angiogenic potency of endothelial cells in the murine ischemic limb suggesting the potential of UCX® as a new therapeutic tool for critical limb ischemia.

Molecular cloning and characterization of ribosomal RNA genes from the brine shrimp.

A library of genomic DNA from the brine shrimp, Artemia, has been constructed with the Charon 4A phage vector, utilizing EcoRI passenger fragments. Screening this library with purified Xenopus laevis cloned rDNA genes has resulted in the identification and plaque purification of a recombinant containing a complete Artemia (18 S + 26 S) rDNA repeat unit. A physical map derived from the analysis of restriction endonuclease digests of the repeat unit, which measures 13.9 kilobase pairs, is similar to the map derived from genomic DNA. In common with several other species, the 26 S rRNA gene terminates with a HindIII recognition site.

Both induction and morphogenesis of cyst nematode feeding cells are mediated by auxin.

Various lines of evidence show that local changes in the auxin concentration are involved in the initiation and directional expansion of syncytia induced by cyst nematodes. Analysis of nematode infections on auxin-insensitive tomato and Arabidopsis mutants revealed various phenotypes ranging from complete inhibition of syncytium development to a decrease in hypertrophy and lateral root formation at the infection site. Specific activation of an auxin-responsive promoter confirmed the role of auxin and pointed at a local accumulation of auxin in developing syncytia Disturbance of auxin gradients by inhibiting polar auxin transport with N-(1-naphthyl)phtalamic acid (NPA) resulted in abnormal feeding cells, which were characterized by extreme galling, massive disordered cell divisions in the cortex, and absence of radial expansion of the syncytium initial toward the vascular bundle. The role of auxin gradients in guiding feeding cell morphogenesis and the cross-talk between auxin and ethylene resulting in a local activation of cell wall degrading enzymes are discussed.

An efficient cDNA-AFLP-based strategy for the identification of putative pathogenicity factors from the potato cyst nematode Globodera rostochiensis.

A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis. Three independent criteria were used for selection. First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages. For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism). Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles. This was checked by in situ hybridization. As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion. Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified. No dorsal gland genes have been cloned before from plant-parasitic nematodes. The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene. Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses. Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.

Naturally induced secretions of the potato cyst nematode co-stimulate the proliferation of both tobacco leaf protoplasts and human peripheral blood mononuclear cells.

Naturally induced secretions from infective juveniles of the potato cyst nematode Globodera rostochiensis co-stimulate the proliferation of tobacco leaf protoplasts in the presence of the synthetic phytohormones alpha-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). With the use of a protoplast-based bioassay, a low-molecular-weight peptide(s) (< 3 kDa) was shown to be responsible for the observed effect. This mitogenic oligopeptide(s) is functionally dissimilar to auxin and cytokinin and, in addition, it does not change the sensitivity of the protoplasts toward these phytohormones. In combination with the mitogen phytohemagglutinin (PHA), cyst nematode secretions also co-stimulated mitogenesis in human peripheral blood mononuclear cells (PBMC). The stimulation of plant cells isolated from nontarget tissue--these nematodes normally invade the roots of potato plants--suggests the activation of a general signal transduction mechanism(s) by an oligopeptide(s) secreted by the nematode. Whether a similar oligopeptide-induced mechanism underlies human PBMC activation remains to be investigated. Reactivation of the cell cycle is a crucial event in feeding cell formation by cyst nematodes. The secretion of a mitogenic low-molecular-weight peptide(s) by infective juveniles of the potato cyst nematode could contribute to the redifferentiation of plant cells into such a feeding cell.