Identifikasjon av fotoreseptorer med funksjonell karakterisering av fotolyase i Pseudoidium neolycopersici

Powdery mildew caused by Pseudoidium neolycopersici is one of the most destructive diseases, occurring predominantly on greenhouse grown tomatoes, and has gained importance worldwide due to severe economic losses in terms of quality and yield. Because of long winter months, the Norwegian tomato production is dependent on protected cultivation systems with controlled environment facilities. Microclimate inside greenhouses provide ideal conditions for development of powdery mildew. The use of resistant cultivars is the first priority for efficient and environmentally friendly management of this disease. However, most of the greenhouse tomato varieties have attained susceptibility to powdery mildew. Therefore, powdery mildew is essentially managed by the use of chemical fungicides. Previous research demonstrated that short wavelength (< 290 nm) ultraviolet radiation (UV) alone or in combination with red light has a practical potential in combating powdery mildew through inhibition of its development. The efficacy of short wavelength UV on germination inhibition of conidia was significantly reduced by UV of longer wavelengths or blue light. This suggested the possible involvement of a light-mediated repair of UV induced DNA damage in powdery mildew. This thesis is based on four articles. In the first article, the effect of short wavelength UV, and subsequent exposure to different incubation wavelengths on conidial germination was assessed to determine the germination recovery spectra of P. neolycopersici. The second article was centered on genome-wide investigation of the presence of photoreceptor genes in three powdery mildew fungi; P. neolycopersici, Podosphaera xanthii, and Podosphaera aphanis. The third article is focused on the identification of the photolyase gene in P. neolycopersici, and its functional characterization. Variation in inhibition and recovery of conidial germination among five isolates of P. neolycopersici treated with similar optical conditions, and a possible explanation for this variation are reported in the fourth article. Petri dish level experiments with conidia of P. neolycopersici were conducted in controlled environment chambers to examine i) the wavelength and dose dependent efficacy of UV (254 nm to 313 nm) on inhibition of conidial germination, ii) the recovery action potential of optical radiation (310 nm to 730 nm) applied subsequently to inhibitory UV treatment, and iii) the lapse time between inhibitory UV treatment, and subsequent exposure to recovery wavelength (peak at 454 nm). After treatments with UV at 254 nm or 283 nm for 30 s followed by dark incubation, less than 10% of the conidia germinated compared to 100% in non-UV controls. Conidial germination was almost negligible if the exposure duration increased to 4 min. With broad spectrum UV (peak 313 nm) conidial germination was about 60% after 1 min of exposure, and about 35% after 2 to 4 min of exposure followed by subsequent dark incubation. UV of 310 nm was ineffective in conidial germination inhibition when applied for ≤ 4 min. Germination of conidia treated with inhibitory UV (254 nm, 283 nm, or broad spectrum UV with peak at 313 nm) was significantly higher (above 73%) if incubated subsequently in optical radiation within the 350 nm to 500 nm range (germination recovery) than if incubated in darkness (control). Furthermore, germination recovery depended on the characteristics of the inhibitory UV treatment (wavelength, and duration of exposure) and the lapse time between inhibitory UV treatment, and subsequent exposure to optical radiation in the recovery range. These findings indicated the presence of photoreceptors in powdery mildew fungi with light mediated UV damage recovery function. Draft genomes of P. neolycopersici, P. xanthii, and P. aphanis, causal agents of powdery mildews of tomato, cucumber and strawberry respectively, were assembled from next generation high throughput sequence data and annotated. An in-depth sequence analysis of these draft genomes revealed the presence of genes similar to all major classes of photoreceptor genes including photolyase, cryptochrome, white collar, phototropin, phytochrome, and UVR8. Three genes were identified as similar to blue light photoreceptors of the cryptochrome/photolyase family (CPF). Photolyase mutant Escherichia coli cells were transformed separately with each of the three CPF-like genes identified in P. neolycopersici, and exposed to brief UV with peak at 254 nm followed by blue light or by darkness (control). One of the three putative CPF-like genes showed photoreactivation activity with cell survival rate of > 98% under blue light compared to the control, and was therefore confirmed as a functional photolyase. Phylogenetic analysis showed that it belonged to class I cyclobutane pyrimidine dimer (CPD) photolyases. The in vitro action spectra of this photolyase was between 365 nm and 454 nm. This perfectly coincides with the conidia germination recovery spectra of P. neolycopersici. Quantitative RT-PCR suggested that the expression of the photolyase gene in P. neolycopersici conidia was significantly induced by brief UV treatment (peak at 254 nm). The expression level was maximum at 4 h after UV treatment (peak at 254 nm) under dark incubation. Photolyase gene expression was significantly reduced in UV (peak at 254 nm) treated conidia when subsequently incubated in red light. The effect of similar doses of UV treatment (peak at 254 nm) followed by incubation with dark or blue light on conidial germination was studied in five isolates of P. neolycopersici collected from different regions in Norway and the Netherlands. Conidia were assessed for germination 24 h after incubation. The results showed variation in the effect of UV on germination and germination recovery under blue light incubation after UV treatment. Two isolates showed more germination recovery compared to the other three isolates. Genomic DNA from these five isolates were extracted, and the photolyase gene was PCR amplified by primer walking. Amplified PCR fragments were sequenced by Sanger sequencing and reads were analyzed for variation. The photolyase gene showed no variation among the five isolates. The findings of this study provide application strategies for designing optical based management programs against powdery mildews by appropriate selection and combination of wavelengths with application time and duration.Meldugg forårsaket av Pseudoidium neolycopersici av er en av de alvorligste sykdommene på tomater og gir store skader i mange land. På grunn av den lange vinteren foregår norsk tomatproduksjon i veksthus, og mikroklimaet i veksthus gir ideelle forhold for meldugg. Bruk av resistente sorter er den mest effektive og miljøvennlige måten å kontrollere sykdommen på. De fleste tomatsortene brukt i veksthus er mottakelige for meldugg, og derfor blir det brukt kjemiske midler til bekjempelse. Det er tidligere vist at kortbølget (< 290 nm) ultrafiolett lys (UV) brukt alene eller i kombinasjon med rødt lys er effektivt som tiltak mot meldugg. Effekten av slik UV-behandling på spiring av konidiesporer av melduggsoppen ble signifikant redusert av UV av høyere bølgelengder og blått lys. Dette indikerer at det skjer en lys-påvirket reparasjon av skaden som UV ved lavere bølgelengder har påført soppen. Denne avhandlingen er basert på fire artikler. I den første ble effekten av kortbølge-UV med påfølgende eksponering for andre bølgelengder undersøkt for å finne lysspekteret som gjenoppretter sporespiring hos P. neolycopersici. Den andre artikkelen er et studium av forekomst av fotoreseptor-gener i genomene til tre melduggsopper; P. neolycopersici, P. xanthii og P. aphanis. Den tredje artikkelen er fokusert på identifisering av fotolyase-genet hos P. neolycopersici og karakterisering av genets funksjon. Variasjon i inhibering og reparasjon av sporespiring blant fem isolat av P. neolycopersici behandlet under de samme optiske forholdene og mulig forklaring på denne variasjonen er rapportert i den fjerde artikkelen. Forsøk i Petri-skåler ble gjennomført under kontrollerte forhold i vekstrom for å undersøke i) bølgelengde- og doseavhengig effekt av behandling med kortbølge-UV (254 – 313 nm) på spiring av konidiesporer, ii) aksjonspotensialet for gjenoppretting av spiring ved hjelp av optisk stråling (310 – 730 nm) tilført etter kortbølge-UV, iii) effekt av lengde på tidsrom mellom behandling med kortbølge-UV og behandling med bølgelengder som gjenoppretter spiring (topp ved 454 nm). Mindre enn 10% av sporene spirte etter 30 sekunders eksponering ved 254 eller 283 nm og påfølgende inkubering i mørket, sammenlignet med 100% for kontrollen. Det var ingen spiring hvis eksponeringstiden økte til 4 minutter. Ved bruk av en bredspektret UV-lampe (topp på 313 nm) var sporespiringen respektive 60 og 35% sammenlignet med ubehandlet kontroll ved eksponering i 1 og 2 – 4 minutt. UV på 310 nm hadde ingen spirehemmende effekt når det ble påført i ≤ 4 min. Hos konidiesporer som var behandlet med kortbølge-UV (254, 283 eller bredt spekter med topp på 313 nm) etterfulgt av eksponering ved bølgelengder mellom 350 og 500 nm (gjenoppretting), var spireprosenten signifikant høyere (73%) enn hos de som ble inkubert i mørke etter behandlingen med kortbølge-UV (kontroll). Videre var gjenoppretting av sporespiring etter behandling med kortbølge-UV avhengig av bølgelengde og lengde av UV-behandlingen og hvor langt tidsrommet er mellom UV-behandling og påfølgende lyseksponering var. Disse funnene indikerer tilstedeværelse av fotoreseptorer hos melduggsopper med lysregulert reparasjon av UV-skade. Utkast til genomsekvenser for P. neolycopersici, P. xanthii og P. aphanis (patogener på respektive tomat, agurk og jordbær) basert på neste-generasjons-sekvensering ble satt sammen og annotert. En grundig analyse av genomene påviste gener i alle hovedklassene av fotoreseptor-gener, inkludert fotolyase, kryptokrom, white collar, fototropin, fytokrom og UVR8. Tre gener ble identifisert som fotoreseptorer for blått lys i kryptokrom/fotolyase superfamilien (CPF). Hvert av de tre CPF-lignende genene hos P. neolycopersici ble enkeltvis klonet i en fotolyase-mutant av Escherichia coli. Mutantene ble eksponert for kortvarig UV med topp på 254 nm etterfulgt av blått lys eller inkubering i mørke (kontroll). Overlevelsesraten etter UV-behandlingen indikerte at det blant de tre CPF-lignende genene var ett funksjonelt fotolyase-gen. Dette genet ga en overlevelse på over 98% etter behandling med blått lys sammenlignet med kontrollen. Fylogenetisk analyse viste at genet tilhørte klasse I cyclobutane pyrimidine dimer (CPD) fotolyase. Aksjonsspekteret ble vist in vitro å være fra 365 til 454 nm, noe som stemmer helt med lysspekteret for gjenoppretting av sporespiring hos UV-skadet P. neolycopersici. Kvantitativ RT-PCR viste at uttrykket av fotolyase-genet hos konidiesporer av P. neolycopersici ble indusert av kort behandling med UV (topp på 254 nm). Uttrykket var maksimalt etter 4 timer i påfølgende mørke, og rødt lys etter behandlingen med UV (topp på 254 nm) reduserte uttrykket. Fem isolater av P. neolycopersici ble testet for sporespiring etter behandling med UV (topp på 254 nm) etterfulgt av inkubasjon i mørke eller i blått lys. Isolatene var fra Norge og Nederland. Det var en variasjon i effekten av UV på spirehemming og i hvor stor grad blått lys gjenopprettet spiringen. To av isolatene responderte sterkere enn de tre andre. Genomisk DNA fra de fem isolatene ble ekstrahert, og fotolyase-genet ble PCR-amplifisert ved «primer walking». Amplifiserte PCR-fragment ble sekvensert med Sanger-sekvensering og analysert for variasjoner. Det var ingen variasjon i fotolyase-genet blant de fem isolatene. Funnene i dette studiet gir oss viktige kriterier for bedre design av optisk-basert behandling mot meldugg ved riktig valg av bølgelengder, behandlingstidspunkt og -varighet

Variation in UV-mediated damage recovery among Pseudoidium neolycopersici isolates: possible mechanisms.

publishedVersio

Noncompliance within the pharmaceutical industry: A phenomenological case study

Pharmaceutical companies endeavor to manufacture safe and effective products in an increasingly competitive environment. In their anxiety to compete, pharmaceutical companies compromise on safety, resulting in product recall. In 1995, 91 prescription drugs were recalled, this number rose to 401 in 2005, an increase of 200%. Additionally, in 2005, 101 non-prescription drugs were recalled as compared to 61 in 1995, a 66% increase. The primary reason for these recalls was lack of meeting the regulation, specifically the Good Manufacturing Practices. The Food and Drug Administration (FDA) reported that lack of adherence to procedures was the most repeated citation received by pharmaceutical companies. This qualitative phenomenological case study utilized a modified van Kaam method by Moustakas using in-depth, semi-structured interviews to explore the perceptions of a purposive sampling of pharmaceutical managers who have been responsible for achieving and maintaining compliance. The study obtained their perceptions and lived experiences in the challenges that they faced in trying to achieve and maintain compliance. The study also utilized archival data to examine the trends of deficiencies cited by the FDA. Data suggested that the challenges arose from inadequate processes, poorly trained employees, poor drug development, time pressures, and improper documentation. Data also suggested that staff would emulate the pharmaceutical leader about compliance. If the leader is committed to compliance, staff will follow through with compliant practices

Identification of photoreceptors with functional characterization of photolyase in Pseudoidium neolycopersici

Powdery mildew caused by Pseudoidium neolycopersici is one of the most destructive diseases, occurring predominantly on greenhouse grown tomatoes, and has gained importance worldwide due to severe economic losses in terms of quality and yield. Because of long winter months, the Norwegian tomato production is dependent on protected cultivation systems with controlled environment facilities. Microclimate inside greenhouses provide ideal conditions for development of powdery mildew. The use of resistant cultivars is the first priority for efficient and environmentally friendly management of this disease. However, most of the greenhouse tomato varieties have attained susceptibility to powdery mildew. Therefore, powdery mildew is essentially managed by the use of chemical fungicides. Previous research demonstrated that short wavelength (< 290 nm) ultraviolet radiation (UV) alone or in combination with red light has a practical potential in combating powdery mildew through inhibition of its development. The efficacy of short wavelength UV on germination inhibition of conidia was significantly reduced by UV of longer wavelengths or blue light. This suggested the possible involvement of a light-mediated repair of UV induced DNA damage in powdery mildew. This thesis is based on four articles. In the first article, the effect of short wavelength UV, and subsequent exposure to different incubation wavelengths on conidial germination was assessed to determine the germination recovery spectra of P. neolycopersici. The second article was centered on genome-wide investigation of the presence of photoreceptor genes in three powdery mildew fungi; P. neolycopersici, Podosphaera xanthii, and Podosphaera aphanis. The third article is focused on the identification of the photolyase gene in P. neolycopersici, and its functional characterization. Variation in inhibition and recovery of conidial germination among five isolates of P. neolycopersici treated with similar optical conditions, and a possible explanation for this variation are reported in the fourth article. Petri dish level experiments with conidia of P. neolycopersici were conducted in controlled environment chambers to examine i) the wavelength and dose dependent efficacy of UV (254 nm to 313 nm) on inhibition of conidial germination, ii) the recovery action potential of optical radiation (310 nm to 730 nm) applied subsequently to inhibitory UV treatment, and iii) the lapse time between inhibitory UV treatment, and subsequent exposure to recovery wavelength (peak at 454 nm). After treatments with UV at 254 nm or 283 nm for 30 s followed by dark incubation, less than 10% of the conidia germinated compared to 100% in non-UV controls. Conidial germination was almost negligible if the exposure duration increased to 4 min. With broad spectrum UV (peak 313 nm) conidial germination was about 60% after 1 min of exposure, and about 35% after 2 to 4 min of exposure followed by subsequent dark incubation. UV of 310 nm was ineffective in conidial germination inhibition when applied for ≤ 4 min. Germination of conidia treated with inhibitory UV (254 nm, 283 nm, or broad spectrum UV with peak at 313 nm) was significantly higher (above 73%) if incubated subsequently in optical radiation within the 350 nm to 500 nm range (germination recovery) than if incubated in darkness (control). Furthermore, germination recovery depended on the characteristics of the inhibitory UV treatment (wavelength, and duration of exposure) and the lapse time between inhibitory UV treatment, and subsequent exposure to optical radiation in the recovery range. These findings indicated the presence of photoreceptors in powdery mildew fungi with light mediated UV damage recovery function. Draft genomes of P. neolycopersici, P. xanthii, and P. aphanis, causal agents of powdery mildews of tomato, cucumber and strawberry respectively, were assembled from next generation high throughput sequence data and annotated. An in-depth sequence analysis of these draft genomes revealed the presence of genes similar to all major classes of photoreceptor genes including photolyase, cryptochrome, white collar, phototropin, phytochrome, and UVR8. Three genes were identified as similar to blue light photoreceptors of the cryptochrome/photolyase family (CPF). Photolyase mutant Escherichia coli cells were transformed separately with each of the three CPF-like genes identified in P. neolycopersici, and exposed to brief UV with peak at 254 nm followed by blue light or by darkness (control). One of the three putative CPF-like genes showed photoreactivation activity with cell survival rate of > 98% under blue light compared to the control, and was therefore confirmed as a functional photolyase. Phylogenetic analysis showed that it belonged to class I cyclobutane pyrimidine dimer (CPD) photolyases. The in vitro action spectra of this photolyase was between 365 nm and 454 nm. This perfectly coincides with the conidia germination recovery spectra of P. neolycopersici. Quantitative RT-PCR suggested that the expression of the photolyase gene in P. neolycopersici conidia was significantly induced by brief UV treatment (peak at 254 nm). The expression level was maximum at 4 h after UV treatment (peak at 254 nm) under dark incubation. Photolyase gene expression was significantly reduced in UV (peak at 254 nm) treated conidia when subsequently incubated in red light. The effect of similar doses of UV treatment (peak at 254 nm) followed by incubation with dark or blue light on conidial germination was studied in five isolates of P. neolycopersici collected from different regions in Norway and the Netherlands. Conidia were assessed for germination 24 h after incubation. The results showed variation in the effect of UV on germination and germination recovery under blue light incubation after UV treatment. Two isolates showed more germination recovery compared to the other three isolates. Genomic DNA from these five isolates were extracted, and the photolyase gene was PCR amplified by primer walking. Amplified PCR fragments were sequenced by Sanger sequencing and reads were analyzed for variation. The photolyase gene showed no variation among the five isolates. The findings of this study provide application strategies for designing optical based management programs against powdery mildews by appropriate selection and combination of wavelengths with application time and duration

Variation in UV-mediated damage recovery among Pseudoidium neolycopersici isolates: possible mechanisms.

publishedVersio

Protocol for efficient opportunistic communication

In typical wireless networks, end-to-end routing is a usual way to deliver data packets from source nodes to destination nodes. In the case of link failures when no alternative route is found, the routing protocols will drop these packets. As a way to improve the packet delivery ratio, an integration of the store-carry-forward features with the traditional end-to-end communication has been already proposed. The existing solutions propose one-time only switching from one communication mode to another should the link failures occur. In this paper, we propose a hybrid protocol to support the dynamic switch between the two modes of communication should the link conditions changed. That is, the protocol utilises the ability to buffer packets when end-to-end routes are not possible, and leverages the end-to-end routes whenever they become available to ensure performance. We evaluate the proposed protocol using a set of comprehensive simulation scenarios to systematically demonstrate its significant improvement in packet delivery over one of the best representative routing protocol for end-to-end routing

A Prospective Study to assess the quality of life among the patients of Generalized Tonic Clonic Seizure receiving treatment phenytoin plus clobazam Versus Sodium Valproate plus clobazam therapy

Background: Epilepsy is common neurological abnormality affecting a large population. It is recommended to begin treatment of epilepsy with antiepileptic drugs (AEDs) as soon as possible. Fear, misunderstanding, discrimination and social stigma have surrounded epilepsy for centuries, this stigma is still continues in many countries and can impact on the quality of life for people with this disease and their families. Aims and Objective: To compare effect of phenytoin plus clobazam (Group 1) and sodium valproate plus clobazam therapy (Group 2) on quality of life among GTCS patients attending neurology out-patient department at tertiary care center in Gwalior district. Materials and Methods:&nbsp; Present study was prospective study conducted between period of February 2019 to January 2020 on 45-45 patients each receiving either phenytoin plus clobazam or sodium valproate plus clobazam dual therapy for the treatment of patients with Generalized tonic clonic seizures. Frequency , percentage ,&nbsp; mean ,&nbsp; standard deviation , independent student t test , Chi square test , one way repeated measures of ANOVA was applied, Bonferroni Adjustment calculated by using SPSS ver.20 software. P value &lt;0.05 was considered to be statistical significant. Results: &nbsp;For Question 1, in group-1 mean score was 3.29±0.45 at 0 month (baseline), which reduced to 2.51±0.50 and 2.11±0.48 at 3 and 6 months. In group-2 mean score was 3.31±0.46 at 0 month, which reduced to 2.35±0.48 and 1.95±0.29 at 3 and 6 months. For Question 2, in group-1 mean score was 3.44±0.54 at 0 month (baseline), which reduced to2.55±0.54 and 2.22±0.51 at 3 and 6 months. In group-2 mean score was 3.64±0.48 at 0 month, which reduced to 2.77±0.55 and 2.44±0.54 at 3 and 6 months. There was statistically significant improvement was seen within the group at 3 and 6 month as compare to baseline in both groups for question 1, question 2, question 3, question 4;&nbsp; P&lt;0.01). Conclusion: It can be concluded that both combinations of our study groups have better impact on quality of life from baseline till the end of study. But on comparison between them, we have not found statistically significant difference. &nbsp

A performance study of hybrid protocols for opportunistic communications

Typically, many multi-hop wireless networks rely on routing protocols to form end-to-end routes to deliver data packets from the sources to the respective destinations. In the case of link failures occur due to interference or node mobility, these protocols will try to reroute. When no alternative route is found, packets will be dropped as a result. As a way to improve the packet delivery ratio, these protocols can be extended to support the store-carry-forward features. We propose a hybrid approach to enable dynamic switching between mode of communication according to the link conditions. This is different from other existing solutions where the switching occur only when the end-to-end route fails, and packets will be delivered using another communication mode for the rest of the routing path. In this paper, we present the implementation of incorporating the dynamic switching into two routing protocols (AODV and OLSR) and conduct a performance study for the two hybrid protocols in a number of simulation based scenarios

Functional characterization of <i>Pseudoidium neolycopersici</i> photolyase reveals mechanisms behind the efficacy of nighttime UV on powdery mildew suppression

Powdery mildews can be controlled by brief exposure to ultraviolet (UV) radiation with devastating effect on their developmental stages including conidia germination. The treatment effect can be impaired by subsequent exposure to UV-A/blue light. UV-A/blue light-activated photolyase may be responsible for this and therefore we tested the function of three cryptochrome/photolyase family (CPF)-like genes (OINE01015670_T110144, OINE01000912_T103440, and OINE01005061_T102555) identified in the obligate biotrophic fungus Pseudoidium neolycopersici, the cause of tomato powdery mildew. A photolyase-deficient mutant of Escherichia coli transformed with coding sequence of OINE01000912_T103440 and exposed to brief (UV)-C treatment (peak emission at 254 nm) showed photoreactivation and cell survival when exposed to subsequent blue light, indicating complementation of photolyase activity. In contrast, the same photolyase-deficient E. coli transformed with the coding sequences of other two CPF-like genes did not survive this treatment, even though their expression were confirmed at protein level. This confirmed that OINE01000912_T103440 is a gene encoding photolyase, here named PnPHR1, with functionality similar to the native photolyase in E. coli, and classified as a class I cyclobutane pyrimidine dimer (CPD) photolyase. Modeling of the 634-amino acid sequence of PnPHR1 suggested that it is capable of binding flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF). However, spectroscopic data of the protein produced in an E. coli expression system could only reveal the presence of a reduced form of FAD, i.e., FADH– as an intrinsic chromophore. Within the tested wavelength range of 365–525 nm, the survival of photolyase-deficient mutant E. coli transformed with PnPHR1 showed a broad action spectrum from 365 to 454 nm. This was very similar to the previously characterized action spectrum for survival of P. neolycopersici conidia that had been treated with UV-C. Quantitative RT-PCR revealed that the expression of PnPHR1 in P. neolycopersici conidia was induced by UV-C, and peak expression occurred 4 h after brief UV-C treatment. The expression of PnPHR1 was repressed when incubated in red light after the UV-C treatment, but not when incubated in UV-A/blue light. The results may explain why the disease-reducing effect of short wavelength UV is impaired by exposure to UV-A and blue light.publishedVersio