Identification of in vivo Protein Targets of Nitric Oxide in Drosophila melanogaster

Nitric Oxide (NO) is known to alter cell proliferation and growth. This project investigated the molecular targets of NO, using the salivary glands of Drosophila melanogaster third instar larvae. A screen of 75 transgenic Drosophila lines expressing Yellow Fluorescent Protein tagged proteins, for those that showed a rapid alteration in sub-cellular localization after organ culture with an NO donor was undertaken. This screen revealed Mi-2 as a target of NO action. This was confirmed in vivo as localization of Mi-2-YFP was altered in whole salivary glands expressing Nitric Oxide Synthase (NOS2). The localization of the endogenous Mi-2 protein was similarly altered in NOS2 expressing single cells. A quantitative analysis using the image analysis software “Velocity” demonstrated an increase in the nuclear concentration of Mi-2 protein in these cells. Targeted expression of RNAi-Mi-2 as well as analysis of Mi-2 transheterozygous mutant larvae revealed that NO can impart its reduced growth phenotype independently of Mi-2. Dref can associate with Mi-2 and can control cell proliferation and growth. Localisation of Dref did not show any noticeable change in single cells expressing NOS. However when these cells were double labelled both with anti-Dref and anti-Mi-2 antibodies, the anti-Dref staining was altered. This indicates that NO can alter the availability of the Dref antigen by reorganising the Mi-2/Dref complex. Mi-2 is a component of the NuRD complex. The effect of NO in altering the Mi-2/Dref complex was exploited to investigate the effect of simj (a regulatory component of NuRD), on either of the proteins after potential disassociation of the complex. The data demonstrated an independent effect of simj on the localization of each protein. NO and FOXO regulate the expression of a common set of genes and FOXO is required for the anti growth properties of NO. Thus the effect of FOXO expression on Mi-2 and Dref protein distribution was determined. Although no consistent alteration of Mi-2 localization was observed, a quantitative analysis showed a large increase of Dref protein concentration in FOXO expressing cells. Double antibody staining of these cells with both anti-Dref and anti-Mi-2 antibodies showed a novel nuclear localization of the proteins imparted by FOXO. Thus this study has identified the regulation of the Mi-2/Dref protein complex as a possible growth control mechanism by NO and FOX

Insights into the Functions of eIF4E-Binding Motif of VPg in Potato Virus A Infection

The interaction between the viral protein genome-linked (VPg) and eukaryotic initiation factor 4E (eIF4E) or eIF(iso)4E of the host plays a crucial role in potyvirus infection. The VPg of potato virus A (PVA) contains the Tyr-X-X-X-X-Leu-phi (YXXXLΦ) binding motif for eIF(iso)4E. In order to investigate its role in PVA infection, we substituted the conserved tyrosine and leucine residues of the motif with alanine residues in the infectious cDNA of PVA (PVAVPgmut). PVAVPgmut RNA replicated in infiltrated leaves, but RNA accumulation remained low. Systemic infection occurred only if a reversion to wild type PVA occurred. VPg was able to stabilize PVA RNA and enhance the expression of Renilla luciferase (3’RLUC) from the 3’ end of the PVA genome. VPgmut could not support either PVA RNA stabilization or enhanced 3’RLUC expression. The RNA silencing suppressor helper-component proteinase (HCPro) is responsible for the formation of PVA-induced RNA granules (PGs) during infection. While VPgmut increased the number of PG-like foci, the percentage of PVA RNA co-localization with PGs was reduced from 86% to 20%. A testable hypothesis for future studies based on these results is that the binding of eIF(iso)4E to PVA VPg via the YXXXLΦ motif is required for PVA RNA stabilization, as well as the transfer to the RNA silencing suppression pathway and, further, to polysomes for viral protein synthesis

Illuminating The Shadows: Unveiling The Concealed Hazards And Intriguing Links Between Light Pollution And Cancer

Shift workers who work at night have been shown to be more at risk for lung, breast, colorectal, and prostate cancers when exposed to artificial light at night (ALAN). Human cancer has been linked to light pollution\u27s disruption of the circadian cycles. Light deprivation reduces carcinogenesis, but an impairment of pineal gland function caused by exposure to a steady light regimen enhanced carcinogenesis. Due to its capacity to stop the cyclic nightly generation of melatonin, artificial light exposure has been hypothesised to be a risk factor for breast cancer in these industrialised nations. Wonder chemical melatonin, sometimes known as the "hormone of darkness," is thought to have a role in many physiological functions and anomalies, such as the regulation of sleep, circadian rhythms, retinal physiology, seasonal reproductive cycles, immune activity, antioxidation and cancer. Telomerase is impacted by light pollution in a number of ways, including indirectly through melatonin. The expression of Telomerase Reverse Transcriptase (TERT) mRNA is first inhibited by melatonin. Second, by blocking NFκB p50/p65 nucleus translocation and their binding to the promoters of human telomerase reverse transcriptase (hTERT) and inducible nitric oxide synthase (iNOS), melatonin increases the antitumor effect of a drug similar to vemurafenib. This suppresses the expression of iNOS and hTERT

Insights into the Functions of eIF4E-Binding Motif of VPg in Potato Virus A Infection

The interaction between the viral protein genome-linked (VPg) and eukaryotic initiation factor 4E (eIF4E) or eIF(iso)4E of the host plays a crucial role in potyvirus infection. The VPg of potato virus A (PVA) contains the Tyr-X-X-X-X-Leu-phi (YXXXL phi) binding motif for eIF(iso)4E. In order to investigate its role in PVA infection, we substituted the conserved tyrosine and leucine residues of the motif with alanine residues in the infectious cDNA of PVA (PVA(VPgmut)). PVA(VPgmut) RNA replicated in infiltrated leaves, but RNA accumulation remained low. Systemic infection occurred only if a reversion to wild type PVA occurred. VPg was able to stabilize PVA RNA and enhance the expression of Renilla luciferase (3'RLUC) from the 3' end of the PVA genome. VPg(mut) could not support either PVA RNA stabilization or enhanced 3'RLUC expression. The RNA silencing suppressor helper-component proteinase (HCPro) is responsible for the formation of PVA-induced RNA granules (PGs) during infection. While VPg(mut) increased the number of PG-like foci, the percentage of PVA RNA co-localization with PGs was reduced from 86% to 20%. A testable hypothesis for future studies based on these results is that the binding of eIF(iso)4E to PVA VPg via the YXXXL phi motif is required for PVA RNA stabilization, as well as the transfer to the RNA silencing suppression pathway and, further, to polysomes for viral protein synthesis.Peer reviewe

VPg-eIF(iso)4E interaction, coat protein production and virion formation in potato virus A infection

Potato Virus A (PVA), which belongs to the family Potyviridae, is a significant agricultural plant virus that causes crop loss worldwide. Most potyvirus resistance is recessive and occurs due to the loss of interaction between the viral protein genome linked (VPg) and the host eukaryotic initiation factor [eIF4E/(iso)4E]. This interaction has been demonstrated in many cultivated plants that are susceptible to potyviruses. Studies on potyvirus resistance have shown that minute changes in either eIF4E/(iso)4E or VPg can cause the interaction to fail, resulting in the development of viral resistance. However, the detailed mechanisms underlying the significant effects of this interaction during potyvirus infection remain unclear. The central domain of the PVA VPg contains an eIF(iso)4E-binding consensus motif, Tyr-X-X-X-X-Leu-phi (YXXXLΦ). The function of this motif during the VPg–eIF(iso)4E interaction, in the context of PVA infection, was investigated in the present study. The tyrosine and the leucine residues at the binding site were replaced with alanine residues in PVA infectious cDNA (icDNA, PVAVPgmut) and in a VPg expression construct (VPgmut). The results showed that PVAVPgmut was capable of replicating inside the host cell, but overall gene expression remained low, similar to the levels observed for a replication-deficient virus. Systemic infection in PVAVPgmut-infected plants only occurred upon reversion to the wild-type PVA, which occurred in 26% of PVAVPgmut-infected plants by 15 days postinfection. Although VPg typically stabilises viral RNA (vRNA) and 3’ renilla luciferase (RLUC) expression, as published previously, the VPgmut failed to perform these functions. The helper component proteinase (HCPro) induces the generation of PVA-induced granules (PGs) during infection and the assembly of vRNA within these PGs to safeguard vRNA from being silenced. Plants infected with PVAVPgmut showed an increased number of PG-like foci in infected cells as compared to the plants infected with PVAWT. However, in compare to the PVAWT the percentage of PVA RNA colocalising with PGs was significantly low in PVAVPgmut infected leaf samples. The host eIF(iso)4E is thought to bind to the PVA VPg via the YXXXLΦ motif, and this interaction is considered to be essential for PVA RNA stabilisation, the transfer of RNA to the RNA silencing suppression pathway, and RNA translocation to polysomes for viral protein synthesis. In the second study, an alternative mechanism was explored involving the production of large quantities of coat protein (CP), which is a multi-functional protein. Tight control over CP production is necessary, depending on the stage of virus infection. Increased CP concentrations have been shown to represses potyviral gene expression. Therefore, CP concentrations are maintained at low levels during active gene expression in the early infection stage through a host-mediated degradation system. During later infection stages, CP is required at high quantities for the production of a large number of stable particles. The present study showed that ectopically expressed VPg enhances reporter expression more pronouncedly from the 3' side of the genome than from the 5’ side. A similar phenomenon was observed towards the later stages of the infection, in which the 3’ CP cistron and the 3’ reporter cistron were expressed more pronouncedly than the central cylindrical inclusion (CI) cistron and the 5’ reporter cistron. The 3’CP and 3’ reporter protein showed different production/accumulation dynamics than were observed for the rest of the genome. CP expression levels were observed to increase in the presence of overexpressed VPg, during both the early infection stage and towards the later infection stage. This process could represent the mechanism through which potyviruses increase CP production for sufficient virion formation. In the third study of this thesis, whether the stabilisation of CP was necessary for successful virion formation was investigated. These results revealed the function of PVA HCPro during CP stabilisation and virion formation. HCPro was found to be unable to stabilise CP in a virus-free system. A number of additional host and viral factors are necessary to produce stable particles. CP stability by HCPro is unrelated to its capacity for silencing suppression and, therefore, cannot be complemented by other viral silencing suppressors. Together, the findings described in this dissertation revealed important mechanisms that underlie potyvirus recessive resistance and the factors that affect stable virion formation.Potyvirdae heimoon kuuluva perunan A virus (PVA) on merkittävä kasvivirus, koska se aiheuttaa maataloudelle satotappioita maailmanlaajuisesti. Suurimmaksi osaksi tunnettu kestävyys potyviruksia vastaan on resessiivistä ja johtuu siitä, että viruksen genomiin linkittyvän VPg-proteiinin ja isäntäkasvin eukaryoottisen initiaatiofaktori eIF4E/(iso)4E:n välillä esiintyvä vuorovaikutus on estynyt. Kyseinen vuorovaikutus on osoitettu useissa potyviruksella infektoituneessa viljelykasvissa. Potyviruskestävyyttä tutkittaessa on todettu, että hyvin pienetkin muutokset eIF4E/(iso)4E:ssä ja VPg:ssä voivat johtaa infektion epäonnistumiseen ja viruskestävyyden kehittymiseen. Se, mistä tämän vuorovaikutuksen merkittävät vaikutukset täsmällisesti johtuvat molekulaarisen mekanismin tasolla on jäänyt epäselväksi. PVA:n VPg:n aminohapposekvenssin keskellä on eIF(iso)4E:tä sitova konsensusmotiivi, Tyr-X-X-X-X-Leu-phi (YXXXLΦ). Tässä tutkimuksessa selvitettiin VPg–eIF(iso)4E vuorovaikutuksen tehtävää PVA infektiossa. Sitoutumisen konsensusmotiivin tyrosiinin ja leusiinin kodonit vaihdettiin alaniinin kodoneiksi PVA:n infektiivisessä cDNA:ssa (PVAVPgmut) ja VPg:n ekspressiokonstruktissa (VPgmut). Tulokset osoittivat, että PVAVPgmut pystyi replikoitumaan isäntäsolussa, mutta geeniekspressiotaso pysyi matalana, samalla tasolla kuin replikaatiokyvyttömällä PVA mutanttiviruksella. PVAVPgmut mutanttivirus aiheutti systeemisen infektion kasvissa vain silloin, jos se muuntui takaisin villityyppiseksi PVA:ksi. Tämä tapahtui 26%:ssa kasveista 15 päivän sisällä infektioista. Vaikka VPg tyypillisesti stabiloi PVA RNA:ta ja vahvistaa virusgenomin 3’päästä tuotetun Renilla lusiferaasireportterin ilmentymistä, kuten aiemmin on julkaistu, VPgmut ei tähän kyennyt. Viruksen HCPro proteiini aloittaa PVA infektion indusoimien granuloiden (PG) muodostuksen ja virus RNA:n (vRNA) assosioituminen näihin rakenteisiin suojaa sitä geenihiljennykseltä. PVAVPgmut mutanttiviruksella infektoiduissa soluissa PG:n kaltaiset rakenteet lisääntyivät villityyppisellä PVA:lla infektoituihin soluihin verrattuna. Kuitenkin vRNA:n assosioituminen PVAVPgmut infektiossa esiintyviin rakenteisiin oli merkittävästi vähäisempää kuin normaalin infektion aikana. Näyttää siis siltä, että isännän eIF(iso)4E sitoutuu PVA VPg: hen YXXXLΦ-motiivin kautta ja että tämä vuorovaikutus on välttämätön PVA RNA: n stabiloimiseksi, siirtämiseksi RNA hiljennyksen ulottumattomiin ja viemiseksi polysomeille virusproteiinisynteesiä varten. Seuraavassa osajulkaisussa tutkittiin PVA infektioissa useisiin tehtäviin osallistuvan kuoriproteiinin (CP:n) laajamittaiseen kertymiseen vaikuttavia mekanismeja. CP määrän kontrollointi infektiovaiheen vaatimalla tavalla on välttämätöntä. Tiedetään, että korkeat CP määrät tukahduttavat potyviruksen geeniekspression. Siksi isäntäkasvin hajoitussysteemi pitää CP konsentraation matalana viruksen aktiivisen geeniekspression aikana infektion alussa. Myöhemmin infektion edettyä, CP:a tarvitaan suuria määriä viruspartikkeleiden runsaaseen tuottoon. Tutkimuksessa näytettiin, että VPg:n ylimäärä edistää reportteriproteiinin ilmentymistä huomattavasti enemmän silloin, kun se tuotetaan virusgenomin 3’ pään puolelta kuin 5’ puolelta. Samankaltainen ilmiö havaittiin infektion loppua kohden, kun CP:tä ja reportteriproteiinia tuotettiin 3’pään kistroneista voimakkaammin, kuin CI-proteiinia genomin keskellä olevasta kistronista tai reportteriproteiinia genomin 5’ puolella olevasta kistronista. Genomin 3’ puolelta CP:n ja reportteriproteiinin tuoton tai akkumulaation dynamiikka oli siis erilaista kuin muista osista PVA genomia. VPg:n ylimäärä saa aikaan CP:n ilmentymistason kasvua sekä infektion alussa, että lopussa. Siksi on mahdollista, että sen prosessin, jonka avulla partikkeleiden muodostukseen tarvittava CP:n määrä saavutetaan, mekanismi liittyy VPg:n määrän säätelyyn. Tämän väitöskirjan kolmannessa osatyössä tutkittiin, tarvitaanko CP:n stabilsoitumista onnistuneeseen partikkelin tuottoon. Työn tulokset paljastivat, että PVA:n HCPro osallistuu CP:n stabilomiseen ja virionin muodostukseen. HCPro ei kuitenkaan pystynyt stabiloimaan CP:tä itsenäisesti ilman virusinfektiota. Lisäksi, stabiilien partikkeleiden muodostus vaatii monia muitakin isäntäkasvin ja viruksen tekijöitä. HCPro:n stabilioiva vaikutus CP:in ei liity HCPro:n geenihiljennyksen tukahduttajana toimimiseen. Siksi stabilointitehtävää ei voitu komplementoida muiden virusten geenihiljennyksen tukahduttajaproteiinien avulla. Yhteenvetona, tässä väitöskirjassa kuvatut molekulaariset mekanismit toimivat joko potyvirusten resessiivisen resistenssin taustalla tai vaikuttavat normaaliin stabiilien viruspartikkeleiden muodostumiseen

Comparitive Nutritional Analysis Of Sweet Yam Bean (Jicama) Milk With Soya Milk -- A Review

The current research focuses on conducting a comprehensive nutritional comparison between sweet yam bean milk and soy milk. Sweet yam bean, also known as Jicama, is an ancient Mexican root vegetable akin to potatoes or turnips. It is safe for consumption, possesses a slightly sweet taste, and has low sugar content, making it a suitable carbohydrate source for individuals with diabetes or those pursuing a low-sugar diet. On the other hand, soy has been a staple ingredient for thousands of years. The study involved an examination of the proximate composition, mineral content, protein fractions, antinutritional elements, and rotenoids in the seeds of Pachyrhizus erosus, the sweet yam bean. In comparison to other legumes, soy seeds exhibited high levels of proteins, lipids, iron (Fe), and calcium (Ca). The predominant protein fraction was mainly composed of globulins, followed by glutelins. Minimal amounts of antinutritional compounds, such as tannins, hemagglutinating activity, and trypsin inhibitory activity, were detected in the sweet yam bean seeds. By conducting this comprehensive comparative analysis, we aim to provide valuable insights into the nutritional benefits and potential applications of sweet yam bean (Jicama) milk, especially in relation to soy milk, which has been a longstanding nutritional staple

Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens

Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi

Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens

Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi