New evidence of cryptic speciation in the family Longidoridae (Nematoda: Dorylaimida)

Longidorid nematodes comprise more than 500 species, and Longidorus and Xiphinema are the most diversified, prevalent, and cosmopolitan genera within plant-parasitic nematodes. This increases the risk of species misidentification. We conducted an integrative morphometric and genetic study on two longidorid species to elucidate the existence of new cases of cryptic speciation within the genera Longidorus and Xiphinema. Detailed morphological, morphometrical, multivariate, and genetic studies were carried out, and mitochondrial and nuclear haploweb analyses were used to differentiate species within the L. iliturgiensis and X. hispanum complexes. Species delimitation using multivariate and haplonet tools of L. iliturgiensis species complex clearly separated L. tabernensis sp. nov. from L. iliturgiensis and L. indalus, and X. subbaetense sp. nov. from X. hispanum and X. adenohystherum. D2-D3, partial 18S, and partial coxI regions were used for inferring their phylogenetic relationships with other species in each genus. The present study provides new insights into the diversity of Longidorus and Xiphinema species detected in southern Spain, and new evidence of cryptic speciation in both genera. These results support our hypothesis that the biodiversity of Longidoridae in southern Europe is higher than previously supposed and is still not fully clarified.     

Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma

Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pN+ status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.     

Blocking the QB‐binding site of photosystem II by tenuazonic acid,a non–host‐specific toxin of Alternaria alternata,activates singlet oxygen‐mediated and EXECUTER‐dependent signalling in Arabidopsis

Necrotrophic fungal pathogens produce toxic compounds that induce cell death in infected plants. Often, the primary targets of these toxins and the way a plant responds to them are not known. In the present work, the effect of tenuazonic acid (TeA), a non–host‐specific toxin of Alternaria alternata, on Arabidopsis thaliana has been analysed. TeA blocks the Q_B‐binding site at the acceptor side of photosystem II (PSII). As a result, charge recombination at the reaction centre (RC) of PSII is expected to enhance the formation of the excited triplet state of the RC chlorophyll that promotes generation of singlet oxygen (¹O₂). ¹O₂ activates a signalling pathway that depends on the two EXECUTER (EX) proteins EX1 and EX2 and triggers a programmed cell death response. In seedlings treated with TeA at half‐inhibition concentration ¹O₂‐mediated and EX‐dependent signalling is activated as indicated by the rapid and transient up‐regulation of ¹O₂‐responsive genes in wild type, and its suppression in ex1/ex2 mutants. Lesion formation occurs when seedlings are exposed to higher concentrations of TeA for a longer period of time. Under these conditions, the programmed cell death response triggered by ¹O₂‐mediated and EX‐dependent signalling is superimposed by other events that also contribute to lesion formation.     

Inheritance and QTL mapping of glandular trichomes type I density in <Emphasis Type="Italic">Cucumis melo</Emphasis> L.

To determine the genetic basis of the density of glandular trichomes type I in melon, a six-generation family and a recombinant inbred lines (F₇) population have been tested for the character; both genetic groups have been obtained from a cross between the Zimbabwean melon line TGR-1551 (high density of glandular trichomes) and the Spanish cultivar ‘Bola de Oro’ (low density of glandular trichomes). Classic quantitative genetic analysis indicated an oligogenic model for the inheritance of the character with an important additive component and strong epistatic relationships between loci. The quantitative trait locus (QTL) analyses revealed that the character is controlled by one major QTL, tric11, which explained between 23.8 and 58.7 % of its phenotypic variance; other minor QTLs of weaker effect and dependent of the assayed growing conditions were also found to be involved in the character expression. A codominant marker, the microsatellite ECM63, was found to be linked to the major QTL tric11. The validation of this marker in advanced backcrosses confirmed that the TGR-1551 alleles for this QTL increased glandular trichome density. The validation of the detected QTL was also done in other genetic backgrounds, which indicated that ECM63 could be effective in the selection of plants with high density of glandular trichomes type I.     

Unravelling the Biodiversity and Molecular Phylogeny of Needle Nematodes of the Genus Longidorus (Nematoda: Longidoridae) in Olive and a Description of Six New Species

The genus Longidorus includes a remarkable group of invertebrate animals of the phylum Nematoda comprising polyphagous root-ectoparasites of numerous plants including several agricultural crops and trees. Damage is caused by direct feeding on root cells as well as by transmitting nepoviruses that cause disease on those crops. Thus, correct identification of Longidorus species is essential to establish appropriate control measures. We provide the first detailed information on the diversity and distribution of Longidorus species infesting wild and cultivated olive soils in a wide-region in southern Spain that included 159 locations from which 449 sampling sites were analyzed. The present study doubles the known biodiversity of Longidorus species identified in olives by including six new species (Longidorus indalus sp. nov., Longidorus macrodorus sp. nov., Longidorus onubensis sp. nov., Longidorus silvestris sp. nov., Longidorus vallensis sp. nov., and Longidorus wicuolea sp. nov.), two new records for wild and cultivate olives (L. alvegus and L. vineacola), and two additional new records for wild olive (L. intermedius and L. lusitanicus). We also found evidence of some geographic species associations to western (viz. L. alvegus, L. intermedius, L. lusitanicus, L. onubensis sp. nov., L. vineacola, L. vinearum, L. wicuolea sp. nov.) and eastern distributions (viz. L. indalus sp. nov.), while only L. magnus was detected in both areas. We developed a comparative study by considering morphological and morphometrical features together with molecular data from nuclear ribosomal RNA genes (D2–D3 expansion segments of 28S, ITS1, and partial 18S). Results of molecular and phylogenetic analyses confirmed the morphological hypotheses and allowed the delimitation and discrimination of six new species of the genus described herein and four known species. Phylogenetic analyses of Longidorus spp. based on three molecular markers resulted in a general consensus of these species groups, since lineages were maintained for the majority of species. This study represents the most complete phylogenetic analysis for Longidorus species to date.     

Neoadjuvant chemotherapy for carcinoma of the oesophagus and oesophago-gastric junction: a six-year experience

Background

Oesophageal cancer is a major clinical problem with a generally poor prognosis. As a result there has been interest in combining surgery with neoadjuvant chemotherapy to try and improve outcomes, although the current evidence for benefit is inconsistent. We aimed to compare, in a non-randomised study, the post-operative complication rate and short and long-term survival of patients who underwent surgical resection for carcinoma of the oesophagus and types I and II carcinoma of the oesophago-gastric junction with or without neo-adjuvant chemotherapy.

Methods

Details of all resections for oesophageal/junctional (types I and II) adenocarcinoma or squamous cell carcinoma between April 2000 and July 2006 were collected prospectively. Data from patients with T3 and/or N1 disease who underwent either neoadjuvant chemotherapy (NAC) or not (non-NAC) were compared. Data were analysed using Kaplan-Meier plots, Mann-Whitney U-test, Cox Regression modelling, and Chi-squared test with Yates' correction where sample sizes <10.

Results

167 patients were included (89 NAC and 78 non-NAC). The in-hospital post-operative mortality rate of the NAC group (n = 2 deaths; 2.2%) was significantly lower (p = 0.045) than the non-NAC group (n = 6 deaths; 7.7%). Most deaths were due to cardio-respiratory complications; however, there was no significant difference in rates of chest infections, anastomotic leaks, wound infections, re-operations, readmission to ITU or overall complications between the two groups. Although both the two-year survival rate (60.7%) and long-term survival of NAC patients (median survival = 793 days; 95% CI = 390–1196) was greater than non-NAC patients (two-year survival rate = 48.7%; median survival = 554 days; 95% CI = 246–862 respectively), these differences were not statistically significant.

Conclusion

This non-randomised study demonstrated that NAC was associated with a significant reduction in post-operative inpatient mortality rate. Whether this can be explained by a decreased co-morbidity in NAC patients or a protective phenomenon associated with NAC remains unclear. This study also demonstrated a greater two-year survival rate and overall median survival time following NAC but this was not statistically significant.

     

Decoding an olfactory mechanism of kin recognition and inbreeding avoidance in a primate

Background  

Like other vertebrates, primates recognize their relatives, primarily to minimize inbreeding, but also to facilitate nepotism. Although associative, social learning is typically credited for discrimination of familiar kin, discrimination of unfamiliar kin remains unexplained. As sex-biased dispersal in long-lived species cannot consistently prevent encounters between unfamiliar kin, inbreeding remains a threat and mechanisms to avoid it beg explanation. Using a molecular approach that combined analyses of biochemical and microsatellite markers in 17 female and 19 male ring-tailed lemurs (Lemur catta), we describe odor-gene covariance to establish the feasibility of olfactory-mediated kin recognition.     

Forisome dispersion in Vicia faba is triggered by Ca2+ hotspots created by concerted action of diverse Ca2+ channels in sieve elements

Remote-controlled Ca²⁺ influx, elicited by electropotential waves, triggers local signaling cascades in sieve elements and companion cells along the phloem of Vicia faba plants. The stimulus strength seems to be communicated by the rate and duration of Ca²⁺ influx into sieve elements (SEs). The cooperative recruitment of Ca²⁺ channels results in a graded response of forisome culminating in full sieve-tube occlusion. Several lines of evidence are integrated into a model that links the mode and strength of the electropotential waves (EPWs) with forisome dispersion, mediated by transiently enhanced levels of local Ca²⁺ release dependent on both plasma membrane and ER Ca²⁺ channels.Key words: distant injury, electropotential wave, remote sieve tube occlusion, activity of sieve element Ca²⁺ channels, signal cascades, Ca²⁺ hotspots     

Basolateral K channels in an insect epithelium. Channel density, conductance, and block by barium

K channels in the basolateral membrane of insect hindgut were studied using current fluctuation analysis and microelectrodes. Locust recta were mounted in Ussing-type chambers containing Cl-free saline and cyclic AMP (cAMP). A transepithelial K current was induced by raising serosal [K] under short-circuit conditions. Adding Ba to the mucosal (luminal) side under these conditions had no effect; however, serosal Ba reversibly inhibited the short-circuit current (Isc), increased transepithelial resistance (Rt), and added a Lorentzian component to power density spectra of the Isc. A nonlinear relationship between corner frequency and serosal [Ba] was observed, which suggests that the rate constant for Ba association with basolateral channels increased as [Ba] was elevated. Microelectrode experiments revealed that the basolateral membrane hyperpolarized when Ba was added: this change in membrane potential could explain the nonlinearity of the 2 pi fc vs. [Ba] relationship if external Ba sensed about three-quarters of the basolateral membrane field. Conventional microelectrodes were used to determine the correspondence between transepithelially measured current noise and basolateral membrane conductance fluctuations, and ion-sensitive microelectrodes were used to measure intracellular K activity (acK). From the relationship between the net electrochemical potential for K across the basolateral membrane and the single channel current calculated from noise analysis, we estimate that the conductance of basolateral K channels is approximately 60 pS, and that there are approximately 180 million channels per square centimeter of tissue area.