The visible touch: in planta visualization of protein-protein interactions by fluorophore-based methods

Non-invasive fluorophore-based protein interaction assays like fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC, also referred to as "split YFP") have been proven invaluable tools to study protein-protein interactions in living cells. Both methods are now frequently used in the plant sciences and are likely to develop into standard techniques for the identification, verification and in-depth analysis of polypeptide interactions. In this review, we address the individual strengths and weaknesses of both approaches and provide an outlook about new directions and possible future developments for both techniques

Government Policy Ecosystem for Entrepreneurship Development in MSEs Sector

Government Policy Ecosystem is central to the entrepreneurship development in any economy and generally includes Policies relating to government spending, taxation and regulation etc. There are two distinct channels through which government Policy ecosystem impacts the rate of entrepreneurship; the first is through its impact on the quantity and quality of inputs going into the entrepreneurial process and the second is through the impact of Policy on the institutional structure that determines the rules of the game under which the entrepreneurial process unfolds. The present study aims to assess the ‘Government Policy Ecosystem’ existing in the Jammu & Kashmir State towards the overall entrepreneurship development in the Micro and Small Enterprises (MSEs) Sector. The study is based on the response of the representative respondents {existing MSEs Sector entrepreneurs from all the three regions (Jammu, Kashmir and Ladakh) of the state} against the parameters: Policy focus and nature; Taxation and other regulatory Policies; and Policy implementation structure. Findings indicate that to the extent the Policy implementation structure is made proper and more coordination is brought in among the EPAs in implementing the state polIcies , there will be a remarkable entrepreneurship development in the MSEs Sector of the State. Towards the end of the study for the robust entrepreneurship development in Micro and Small Enterprises Sector of the State, on the basis of the findings, certain suggestions have been put forth for the improvement in the existing ‘Government Policy Ecosystem’ for MSEs Sector

Relatório de estágio em farmácia comunitária

Relatório de estágio realizado no âmbito do Mestrado Integrado em Ciências Farmacêuticas, apresentado à Faculdade de Farmácia da Universidade de Coimbr

Combination of Novel Green Fluorescent Protein Mutant TSapphire and DsRed Variant mOrange to Set Up a Versatile in Planta FRET-FLIM Assay1[W]

Förster resonance energy transfer (FRET) measurements based on fluorescence lifetime imaging microscopy (FLIM) are increasingly being used to assess molecular conformations and associations in living systems. Reduction in the excited-state lifetime of the donor fluorophore in the presence of an appropriately positioned acceptor is taken as strong evidence of FRET. Traditionally, cyan fluorescent protein has been widely used as a donor fluorophore in FRET experiments. However, given its photolabile nature, low quantum yield, and multiexponential lifetime, cyan fluorescent protein is far from an ideal donor in FRET imaging. Here, we report the application and use of the TSapphire mutant of green fluorescent protein as an efficient donor to mOrange in FLIM-based FRET imaging in intact plant cells. Using time-correlated single photon counting-FLIM, we show that TSapphire expressed in living plant cells decays with lifetime of 2.93 ± 0.09 ns. Chimerically linked TSapphire and mOrange (with 16-amino acid linker in between) exhibit substantial energy transfer based on the reduction in the lifetime of TSapphire in the presence of the acceptor mOrange. Experiments performed with various genetically and/or biochemically known interacting plant proteins demonstrate the versatility of the FRET-FLIM system presented here in different subcellular compartments tested (cytosol, nucleus, and at plasma membrane). The better spectral overlap with red monomers, higher photostability, and monoexponential lifetime of TSapphire makes it an ideal FRET-FLIM donor to study protein-protein interactions in diverse eukaryotic systems overcoming, in particular, many technical challenges encountered (like autofluorescence of cell walls and fluorescence of pigments associated with photosynthetic apparatus) while studying plant protein dynamics and interactions

Extrahepatic portal vein obstruction and portal vein thrombosis in special situations: Need for a new classification

Extrahepatic portal vein obstruction is a vascular disorder of liver, which results in obstruction and cavernomatous transformation of portal vein with or without the involvement of intrahepatic portal vein, splenic vein, or superior mesenteric vein. Portal vein obstruction due to chronic liver disease, neoplasm, or postsurgery is a separate entity and is not the same as extrahepatic portal vein obstruction. Patients with extrahepatic portal vein obstruction are generally young and belong mostly to Asian countries. It is therefore very important to define portal vein thrombosis as acute or chronic from management point of view. Portal vein thrombosis in certain situations such as liver transplant and postsurgical/liver transplant period is an evolving area and needs extensive research. There is a need for a new classification, which includes all areas of the entity. In the current review, the most recent literature of extrahepatic portal vein obstruction is reviewed and summarized

Interaction of maize Opaque-2 and the transcriptional co-activators GCN5 and ADA2, in the modulation of transcriptional activity

International audienceMaize Opaque-2 (ZmO2), a bZip class transcription factor has been shown to activate the transcription of a series of genes expressed in the maturation phase of endosperm development. Activation requires the presence of one or more enhancer binding sites, which confer the propensity for activation by ZmO2 on heterologous promoters and in heterologous plant cell types, such as tobacco mesophyll protoplasts. The region of ZmO2 required for conferring transcriptional activation has been localised to a stretch of acidic residues in the N-terminal portion of the ZmO2 sequence, which is conserved between O2-related bZip factor sequences. Previously we identified the maize homologues of yeast transcriptional co-activators GCN5 and ADA2 that are implicated in nucleosome modification and transcription. In the present study we have shown that transcriptional modulation by ZmO2 involves the intranuclear interaction of ZmO2 with ZmADA2 and ZmGCN5. Forster resonance energy transfer (FRET) based techniques have enabled us to estimate the intracellular site of these intermolecular interactions. As a functional readout of these intranuclear interactions, we used the ZmO2 responsive maize b-32 promoter to drive the beta-glucuronidase (GUS) in the presence and absence of ZmGCN5 and ZmADA2. Our results suggest that the likely recruitment of ZmADA2 and ZmGCN5 modulates the transactivation of b-32 promoter by ZmO2 and that there may be a competition between ZmGCN5 and ZmO2 for binding to the amino-terminal of ZmADA2. The results may be taken as a paradigm for other processes of transcriptional modulation in planta involving acidic activation domains

Recruitment and interaction dynamics of plant penetration resistance components in a plasma membrane microdomain

Many fungal pathogens must enter plant cells for successful colonization. Barley mildew resistance locus o (Mlo) is required for host cell invasion upon attack by the ascomycete powdery mildew fungus, Blumeria graminis f.sp. hordei, and encodes the founder of a family of heptahelical integral membrane proteins unique to plants. Recessively inherited loss-of-function mutant alleles (mlo) result in effective penetration resistance to all isolates of the biotrophic parasite. We used noninvasive fluorescence-based imaging to show that fluorescently tagged MLO protein becomes redistributed in the plasma membrane (PM) and accumulates beneath fungal appressoria coincident with the initiation of pathogen entry into host cells. Polarized MLO accumulation occurs once upon attack and appears to be independent of actin cytoskeleton function. Likewise, barley ROR2 syntaxin, a genetically defined component of penetration resistance to B. graminis f.sp. hordei, and a subset of predicted PM-resident proteins become redistributed to fungal entry sites. We previously identified calmodulin, a cytoplasmic calcium sensor, as an interactor and positive regulator of MLO activity and demonstrate here by FRET microscopy an increase in MLO/calmodulin FRET around penetration sites coincident with successful host cell entry. Our data provide evidence for the formation of a pathogen-triggered PM microdomain that is reminiscent of membrane microdomains (lipid rafts) induced upon attempted entry of pathogenic bacteria in animal cells

Barley MLO Modulates Actin-Dependent and Actin-Independent Antifungal Defense Pathways at the Cell Periphery1[W][OA]

Cell polarization is a crucial process during plant development, as well as in plant-microbe interactions, and is frequently associated with extensive cytoskeletal rearrangements. In interactions of plants with inappropriate fungal pathogens (so-called non-host interactions), the actin cytoskeleton is thought to contribute to the establishment of effective barriers at the cell periphery against fungal ingress. Here, we impeded actin cytoskeleton function in various types of disease resistance using pharmacological inhibitors and genetic interference via ectopic expression of an actin-depolymerizing factor-encoding gene, ADF. We demonstrate that barley (Hordeum vulgare) epidermal cells require actin cytoskeleton function for basal defense to the appropriate powdery mildew pathogen Blumeria graminis f. sp. hordei and for mlo-mediated resistance at the cell wall, but not for several tested race-specific immune responses. Analysis of non-host resistance to two tested inappropriate powdery mildews, Erysiphe pisi and B. graminis f. sp. tritici, revealed the existence of actin-dependent and actin-independent resistance pathways acting at the cell periphery. These pathways act synergistically and appear to be under negative control by the plasma membrane-resident MLO protein

fagocitna aktivnost i fagocitni indeksi polimorfonuklearnih stanica mlijeka (PMN) kao markeri za dijagnozu i praćenje terapijske i profilaktičke učinkovitosti različitih formulacija antioksidanata u suzbijanju mastitisa goveda

A clinical study was undertaken on bovine mastitis in the Kashmir valley to study the relationship between Phagocytic activity (PA) and the phagocytic index (PI) of milk neutrophils (PMN’s) and bovine mastitis. The PA and PI of milk PMN’s were assessed in mastitic animals and compared with normal, healthy, lactating animals. A significant decrease was found in the values of the PA and PI of milk neutrophils in clinical cases of mastitis as compared to the healthy control group. The utility of anti-oxidants in clinical management of mastitis was measured through the response to treatment with trace minerals such as Cu, Zn, Mn and Se in addition to conventional antibiotic therapy. Two groups of mastitic animals received two therapeutic regimens. Group I animals received antibiotics (Enrofloxacin 6.6 mg/kg BID for 5 days) and a self-formulated trace mineral anti-oxidant mixture at therapeutic doses for 7 days, while Group II animals only received the antibiotic (at the same dose rate and frequency). Therapeutic regimen I proved more efficacious than the therapeutic regimen II in the treatment of clinical mastitis, as evidenced by higher recovery rates, a lower number of mean days required for recovery, and higher PA and PI of the milk PMN’s on day 10 post treatment. For prophylactic studies, 24 healthy lactating cows were divided into two groups of 12 animals each. One group was supplemented with a self-formulated trace mineral anti-oxidant mixture at prophylactic doses for 30 days, which resulted in a significant improvement in udder health status in terms of the SCC, PA and PI of milk PMN’s. Supplementation with the antioxidant formulation played a significant role in early recovery and prophylaxis of bovine mastitis. Assessment of PA and PI of milk PMN’s could be utilised as an effective tool for diagnosing susceptibility to the occurrence of mastitis, as well as monitoring the therapeutic efficacy of different treatment regimes.Kliničko istraživanje mastitisa goveda u dolini Kashmir provedeno je kako bi se procijenila povezanost fagocitne aktivnosti (PA) i fagocitnih indeksa (PI) neutrofila u mlijeku (PMN’s) i mastitisa goveda. PA i PI neutrofila u mlijeku analizirani su u životinja s mastitisom i uspoređeni sa zdravim životinjama u laktaciji. Pronađeno je znakovito smanjenje PA i PI neutrofila u mlijeku u slučajevima kliničkog mastitisa u usporedbi s kontrolnom, zdravom skupinom. Korisnost antioksidansa u slučajevima kliničkog mastitisa mjerena je putem odgovora na liječenje mineralima u tragovima kao što su Cu, Zn, Mn i Se, koji su dodani konvencionalnoj antibiotskoj terapiji. Dvije skupine životinja s mastitisom imale su dva terapijska programa. Skupina 1 primala je antibiotik (enrofloksacin 6,6 mg/kg, 2 puta dnevno, tijekom 5 dana) i vlastito pripremljenu mješavinu antioksidantnih minerala u tragovima u terapijskoj dozi tijekom 7 dana, dok je skupina 2 dobivala samo antibiotike (u istoj dozi i intervalu). Terapijski program u skupini 1 pokazao se učinkovitijim od onoga u skupini 2 u liječenju kliničkog mastitisa, što dokazuju veće stope oporavka, manji prosječan broj dana potreban za oporavak te povećane vrijednosti PA i PI polimorfonuklearnih stanica mlijeka deseti dan poslije tretmana. Za istraživanje profilakse 24 zdrave krave u laktaciji podijeljene su u dvije skupine sa po 12 životinja. Jedna je skupina dobivala vlastito pripremljenu mješavinu antioksidantnih minerala u tragovima u profilaktičkoj dozi tijekom 30 dana. To je rezultiralo znakovitim poboljšanjem zdravlja vimena što se vidjelo po vrijednostima SCC, PA i PI neutrofila u mlijeku. Dodatak antioksidantne formulacije znakovito je utjecao na rani oporavak i profilaksu mastitisa u goveda. Procjena PA i PI polimorfonuklearnih stanica mlijeka mogla bi biti učinkovit alat pri dijagnostici sumnje na mastitis kao i praćenju učinkovitosti različitih terapijskih program