Sampling and PCR method for detecting pathogenic Fusarium oxysporum strains in onion harvest

Fusarium basal rot is a worldwide disease problem in onions, and causes substantial losses in onion production, both during the growing season and in the storage. To minimize the post-harvest losses, a protocol for screening of latent infections with pathogenic Fusarium oxysporum strains from harvested onions was developed. This protocol is based on a dual PCR test with primers specific for the fungal species and new SIX3 primers specific for the onion-pathogenic F. oxysporum strains. A pooled sample containing pieces from 50 harvested symptomless onions was prepared for the dual PCR using microwave disruption of the filamentous Fusarium fungi and Whatman FTA(TM) filter paper matrix technology, or as a reference protocol, by extracting DNA with a commercial kit. The two sample preparation protocols gave consistent results with the tested onion samples. Detection limit of the dual PCR protocol was 100 pg of F. oxysporum DNA, in a mixture with onion DNA, when the FTA card was applied. The new protocol reported here is simple and sensitive enough for routine testing, enabling the detection of latent infections in harvest lots even at the infection levels under 10%. Significance and Impact of the Study Fusarium basal rot causes serious problems in onion production. To minimize post-harvest losses, a simple protocol based on FTA(TM) technology and a dual PCR test with Fusarium oxysporum species-specific and pathogenicity-specific primers was developed. By testing pooled onion samples using this method, latent infections with F. oxysporum can be screened from a representative sample of the harvest. This screening method could be a useful tool to manage the post-harvest losses caused by latent infections with F. oxysporum and, with modification of the PCR protocol, with other Fusarium species pathogenic to onion.Peer reviewe

Elevated human placental heat shock protein 5 is associated with spontaneous preterm birth

Background: Specific heat shock proteins are associated with pregnancy complications, including spontaneous preterm birth (SPTB). Placental proteomics and whole exome sequencing recently suggested an association between heat shock protein HSPA5 and uncomplicated SPTB. In the present study, we investigated the localization of and possible roles for HSPA5 in SPTB. Methods: Western blot was performed to validate the result from the previously published proteomic analysis. We used qPCR to assess mRNA expression of genes and immunohistochemistry and immunoelectron microscopy to examine localization of HSPA5 in placental tissue. We silenced the HSPA5 gene in the HTR8/SVneo human trophoblast cell line to investigate possible functions of HSPA5. Results: HSPA5 was upregulated in placentas from SPTBs compared to spontaneous term births. We did not observe upregulation of HSPA5 mRNA in placental samples. The protein was localized in placental trophoblast in both spontaneous preterm and term placentas. Gene silencing of HSPA5 in human trophoblast cell culture affected the inflammatory response and decreased the expression of several proinflammatory genes. Conclusions: We suggest that upregulation of HSPA5 in the placenta is associated with spontaneous preterm labor. HSPA5 may promote the inflammatory response and alter the anti-inflammatory state of the placenta which could eventually lead to premature labor. Impact: We validated upregulation of HSPA5 in placentas from spontaneous preterm birth. HSPA5 was not upregulated at transcriptional level which suggests that it may be regulated post-translationally.Silencing HSPA5 in a human trophoblast–derived cell line suggested that HSPA5 promotes expression of proinflammatory cytokines. The emerging inflammation could lead to spontaneous preterm labor.Identifying inflammatory pathways and factors associated with spontaneous preterm birth increases knowledge of the molecular mechanisms of premature labor. This could provide cues to predict imminent premature labor and lead to information about how to safely maintain pregnancies.publishedVersionPeer reviewe

Human placental proteomics and exon variant studies link AAT/SERPINA1 with spontaneous preterm birth

Background: Preterm birth is defined as live birth before 37 completed weeks of pregnancy, and it is a major problem worldwide. The molecular mechanisms that lead to onset of spontaneous preterm birth are incompletely understood. Prediction and evaluation of the risk of preterm birth is challenging as there is a lack of accurate biomarkers. In this study, our aim was to identify placental proteins that associate with spontaneous preterm birth. Methods: We analyzed the proteomes from placentas to identify proteins that associate with both gestational age and spontaneous labor. Next, rare and potentially damaging gene variants of the identified protein candidates were sought for from our whole exome sequencing data. Further experiments we performed on placental samples and placenta-associated cells to explore the location and function of the spontaneous preterm labor-associated proteins in placentas. Results: Exome sequencing data revealed rare damaging variants in SERPINA1 in families with recurrent spontaneous preterm deliveries. Protein and mRNA levels of alpha-1 antitrypsin/SERPINA1 from the maternal side of the placenta were downregulated in spontaneous preterm births. Alpha-1 antitrypsin was expressed by villous trophoblasts in the placenta, and immunoelectron microscopy showed localization in decidual fibrinoid deposits in association with specific extracellular proteins. siRNA knockdown in trophoblast-derived HTR8/SVneo cells revealed that SERPINA1 had a marked effect on regulation of the actin cytoskeleton pathway, Slit–Robo signaling, and extracellular matrix organization. Conclusions: Alpha-1 antitrypsin is a protease inhibitor. We propose that loss of the protease inhibition effects of alpha-1 antitrypsin renders structures critical to maintaining pregnancy susceptible to proteases and inflammatory activation. This may lead to spontaneous premature birth.publishedVersionPeer reviewe

Expression of CPPED1 in human trophoblasts is associated with timing of term birth

Understanding of timing of human parturition is incomplete. Therefore, we carried out proteomic analyses of full-term placentas from uncomplicated pregnancies to identify protein signatures associated with the onset of spontaneous delivery. We found quantitative associations of 10 proteins with spontaneous term birth, evident either in the basal or in the chorionic plates or in both. Additional 18 proteins were associated according to the location within placenta indicating local variations in protein amounts. Calcineurin-like phosphoesterase domain-containing 1 (CPPED1), a phosphatase previously suggested dephosphorylating AKT1/PKB, was one of the identified proteins. qRT-PCR revealed the mRNA level of CPPED1 was higher in elective caesarean deliveries than in spontaneous births, while immunohistochemistry showed CPPED1 in cytotrophoblasts, syncytiotrophoblasts and extravillous trophoblasts. Noteworthy, phosphorylation status of AKT1 did not differ between placentas from elective caesarean and spontaneous deliveries. Additionally, analyses of samples from infants indicated that single-nucleotide polymorphisms rs11643593 and rs8048866 of CPPED1 were associated with duration of term pregnancy. Finally, post-transcriptional silencing of CPPED1 in cultured HTR8/SVneo cells by siRNAs affected gene expression in pathways associated with inflammation and blood vessel development. We postulate that functions regulated by CPPED1 in trophoblasts at choriodecidual interphase have a role in the induction of term labour, but it may be independent of AKT1

Conservation of Salmonella Infection Mechanisms in Plants and Animals

Salmonella virulence in animals depends on effectors injected by Type III Secretion Systems (T3SSs). In this report we demonstrate that Salmonella mutants that are unable to deliver effectors are also compromised in infection of Arabidopsis thaliana plants. Transcriptome analysis revealed that in contrast to wild type bacteria, T3SS mutants of Salmonella are compromised in suppressing highly conserved Arabidopsis genes that play a prominent role during Salmonella infection of animals. We also found that Salmonella originating from infected plants are equally virulent for human cells and mice. These results indicate a high degree of conservation in the defense and infection mechanism of animal and plant hosts during Salmonella infection

Expression of αvβ6integrin in oral leukoplakia

The distribution of αvβ6integrin was examined in oral leukoplakia, lichen planus and squamous cell carcinomas using immunohistochemistry. Controls included oral mucosal wounds, chronically inflamed and normal oral mucosa. Integrins β1, β3, β4, β5, fibronectin and tenascin were also studied. The integrin αvβ6was highly expressed throughout the whole lesion of 90% of the squamous cell carcinomas but was not present in any of the normal specimens. αvβ6integrin was also expressed in 41% of the leukoplakia specimens, and 85% of the lichen planus samples, but in none of the tissues with inflammatory hyperplasia or chronic inflammation. The expression of β1 integrins was localized in the basal layer, and that of the β4at the cell surface facing the basement membrane of all specimens. The integrins β3and β5were absent from all normal and leukoplakia specimens. Fibronectin and tenascin were present in the connective tissue underneath the epithelium of all the sections, and their expression was similar in both αvβ6-positive and αvβ6-negative tissues. A group of 28 leukoplakia patients were followed 1–4 years after first diagnosis. In this group, initially αvβ6integrin-positive leukoplakia specimens had high tendency for disease progression while αvβ6-negative specimens did not progress. These results suggest that the expression of αvβ6integrin could be associated in the malignant transformation of oral leukoplakias. © 2000 Cancer Research Campaig

Genomic and neural analysis of the estradiol-synthetic pathway in the zebra finch

<p>Abstract</p> <p>Background</p> <p>Steroids are small molecule hormones derived from cholesterol. Steroids affect many tissues, including the brain. In the zebra finch, estrogenic steroids are particularly interesting because they masculinize the neural circuit that controls singing and their synthesis in the brain is modulated by experience. Here, we analyzed the zebra finch genome assembly to assess the content, conservation, and organization of genes that code for components of the estrogen-synthetic pathway and steroid nuclear receptors. Based on these analyses, we also investigated neural expression of a cholesterol transport protein gene in the context of song neurobiology.</p> <p>Results</p> <p>We present sequence-based analysis of twenty steroid-related genes using the genome assembly and other resources. Generally, zebra finch genes showed high homology to genes in other species. The diversity of steroidogenic enzymes and receptors may be lower in songbirds than in mammals; we were unable to identify all known mammalian isoforms of the 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase families in the zebra finch genome assembly, and not all splice sites described in mammals were identified in the corresponding zebra finch genes. We did identify two factors, Nobox and NR1H2-RXR, that may be important for coordinated transcription of multiple steroid-related genes. We found very little qualitative overlap in predicted transcription factor binding sites in the genes for two cholesterol transport proteins, the 18 kDa cholesterol transport protein (TSPO) and steroidogenic acute regulatory protein (StAR). We therefore performed in situ hybridization for TSPO and found that its mRNA was not always detected in brain regions where StAR and steroidogenic enzymes were previously shown to be expressed. Also, transcription of TSPO, but not StAR, may be regulated by the experience of hearing song.</p> <p>Conclusions</p> <p>The genes required for estradiol synthesis and action are represented in the zebra finch genome assembly, though the complement of steroidogenic genes may be smaller in birds than in mammals. Coordinated transcription of multiple steroidogenic genes is possible, but results were inconsistent with the hypothesis that StAR and TSPO mRNAs are co-regulated. Integration of genomic and neuroanatomical analyses will continue to provide insights into the evolution and function of steroidogenesis in the songbird brain.</p

The Type VI secretion system deploys anti-fungal effectors against microbial competitors

This work was supported by the Wellcome Trust (Senior Research Fellowship in Basic Biomedical Science to S.J.C., 104556; 097377, J.Q.; 101873 & 200208, N.A.R.G.), the MRC (MR/K000111X/1, S.J .C; MC_UU_12016/5, M.T.), and the BBSRC (BB/K016393/1 & BB/P020119/1, J.Q.). We thank Maximilian Fritsch, Mario López Martín and Birte Hollmann for help with strain construction; Gary Eitzen for construction of pGED1; Donna MacCallum for the gift of Candida glabrata ATCC2001; Joachim Morschhäuser for the gift of pNIM1; Gillian Milne (Microscopy and Histology facility, University of Aberdeen) for assistance with TEM; and Peter Taylor, Michael Porter, Laura Monlezun and Colin Rickman for advice and technical assistance.Peer reviewedPostprin

Dielectrophoretic mobility of a spherical particle in 2D hyperbolic quadrupole electrode geometry

Abstract The dielectric properties of material are of importance in various industrial and scientific applications of measurement technology. These properties are usually measured by microwave sensors, methods that provide an average result of the entire sample volume. However, the need to know the dielectric properties of single particles has increased in various segments. Dielectrophoresis (DEP) as a method provides a way to determine dielectric properties, such as permittivity and conductivity, of single particles with good precision by using a sufficiently simple system. In this thesis the DEP platform consists of four electrodes with hyperbolic edge geometry. Geometry produces a linear electric field gradient which leads to a constant DEP force in the active region and ensures fairly straightforward determination of particle properties by means of their DEP mobility. Particle mobility is dependent on particle size, the slope of the electric field gradient, the conductivity of the carrier fluid and the frequency of the electric field. For this thesis has been studied particle characterization on a 2D platform, particle 3D behavior, simultaneous multiphenomena observation and the applicability of transparent indium-tin-oxide (ITO) electrode material to DEP platforms. Experiments were made with polystyrene particles, using carrier fluids of varying conductivity. Electrode transparency enabled holographic 3D imaging and thus also the observation of particle behavior in the depth direction. This 3D imaging revealed the restricted working distance from the electrode plane, as well as the mobility of particles in the depth dimension under the DEP force. It was also observed that there was only a marginal difference between 3D mobility and 2D mobility inside the active region. The dielectric properties of the polystyrene particles were determined and found to differ from those of solid polystyrene, and thus the properties of the carrier fluid have a distinctive effect on particle properties. The measurement achieved good precision in a single particle measurement and thus the total particle consistency could be very low. The main restrictions of the DEP method are the limited working distance and restricted range of particle sizes: based on the results of this thesis, with a fixed size platform the particle size may not vary by more than tenfold.Tiivistelmä Materiaalin dielektriset ominaisuudet ovat merkittäviä monissa teollisissa sekä tieteellisissä mittausteknisissä sovelluksissa. Yleensä nämä ominaisuudet mitataan mikroaaltosensoreilla, joilla mitataan näytetilavuuden keskiarvo. Tarve tietää yksittäisen partikkelin dielektriset ominaisuudet on kuitenkin lisääntynyt useilla segmenteillä. Dielektroforeesi (DEP) on menetelmä, jolla voidaan toistettavasti mitata yksittäisen partikkelin permittiivisyys ja johtavuus suhteellisen yksinkertaisella mittaussysteemillä. Tässä työssä DEP-alusta koostuu neljästä elektrodista, joilla on hyperbelin muotoinen elektrodin reunan muoto. Geometria tuottaa lineaarisen sähkökentän gradientin, mikä johtaa vakio DEP-voimaan alustan aktiivisella alueella. Niinpä partikkelin ominaisuuksien määrittely niiden liikkuvuuden perusteellä on suoraviivaista. Liikkuvuus on riippuvainen partikkelin koosta, sähkökentän gradientin kulmakertoimesta, kantajaliuoksen johtavuudesta sekä käytetyn sähkökentän taajuudesta. Työssä on tutkittu partikkelin karakterisointia 2D-alustalla, partikkelin 3D-käyttäytymistä, samanaikaista moni-ilmiötarkkailua sekä indium-tina-oksidi (ITO) elektrodimateriaalin soveltuvuutta DEP-alustoihin. Kokeet tehtiin polystyreenipartikkeleilla käyttäen eri johtavuuksisia kaliumkloridi-kantajaliuoksia. Elektrodien läpinäkyvyys mahdollisti holografisen 3D-kuvantamisen, jonka avulla havaittiin alustan rajallinen toimintaetäisyys sekä partikkelin syvyyssuuntainen liike DEP-voiman vaikutuksesta. Kuitenkin 3D- ja 2D-liikkeen välillä havaittiin vain vähäinen ero. Määritellyt partikkelin dielektriset ominaisuudet eroavat kiinteän polystyreenin arvoista, jolloin kantajaliuoksen ominaisuuksilla on merkittävä vaikutus määriteltyihin partikkelin ominaisuuksiin. Mittauksilla saavutettiin hyvä toistettavuus jopa yksittäisen partikkelin mittauksista, niinpä näytteen partikkelipitoisuus voi olla erittäin pieni. DEP-menetelmän merkittävimpiä rajoitteita ovat rajallinen toimintaetäisyys sekä rajallinen partikkelikoon variaatio, mikä voi olla yhdellä DEP-alustalla noin kymmenkertainen