Bcl-2 proteins link programmed cell death with growth and morphogenetic adaptations in the fungal plant pathogen Colletotrichum gloeosporioides

Proteins belonging to the Bcl-2 family regulate apoptosis in mammals by controlling mitochondria efflux of cytochrome c and other apoptosis-related proteins. Homologues of human Bcl-2 proteins are found in different metazoan organisms where they play a similar role, while they seem to be absent in plants and fungi. Nonetheless, Bcl-2 protein members can induce or prevent yeast cell death, suggesting that enough functional conservation exists between apoptotic machineries of mammals and fungi. Here we show that induction or prevention of apoptosis by Bcl-2 proteins in the fungal plant pathogen Colletotrichum gloeosporioides is tightly linked with growth and morphogenetic adaptation that occur throughout the entire fungal life cycle. Isolates expressing the pro-apoptotic Bax protein underwent cell death with apoptotic characteristics, and showed alterations in conidial germination that are associated with pathogenic and non-pathogenic life styles. Isolates expressing the anti-apoptotic Bcl-2 protein had prolonged longevity, were protected from Bax-induced cell death, and exhibited enhanced stress resistance. These isolates also had enhanced mycelium and conidia production, and were hyper virulent to host plants. Our findings show that apoptotic-associated machinery regulates morphogenetic switches, which are critical for proper responses and adaptation fungi to different environments.     

Functional Proteomics Identifies Acinus L as a Direct Insulin- and Amino Acid-Dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Substrate

The serine/threonine kinase mammalian target of rapamycin (mTOR) governs growth, metabolism, and aging in response to insulin and amino acids (aa), and is often activated in metabolic disorders and cancer. Much is known about the regulatory signaling network that encompasses mTOR, but surprisingly few direct mTOR substrates have been established to date. To tackle this gap in our knowledge, we took advantage of a combined quantitative phosphoproteomic and interactomic strategy. We analyzed the insulin- and aa-responsive phosphoproteome upon inhibition of the mTOR complex 1 (mTORC1) component raptor, and investigated in parallel the interactome of endogenous mTOR. By overlaying these two datasets, we identified acinus L as a potential novel mTORC1 target. We confirmed acinus L as a direct mTORC1 substrate by co-immunoprecipitation and MS-enhanced kinase assays. Our study delineates a triple proteomics strategy of combined phosphoproteomics, interactomics, and MS-enhanced kinase assays for the de novo-identification of mTOR network components, and provides a rich source of potential novel mTOR interactors and targets for future investigation.The serine/threonine kinase mammalian target of rapamycin (mTOR)¹ is conserved in all eukaryotes from yeast to mammals (1). mTOR is a central controller of cellular growth, whole body metabolism, and aging, and is frequently deregulated in metabolic diseases and cancer (2). Consequently, mTOR as well as its upstream and downstream cues are prime candidates for targeted drug development to alleviate the causes and symptoms of age-related diseases (3, 4). The identification of novel mTOR regulators and effectors thus remains a major goal in biomedical research. A vast body of literature describes a complex signaling network around mTOR. However, our current comparatively detailed knowledge of mTOR''s upstream cues contrasts with a rather limited set of known direct mTOR substrates.mTOR exists in two structurally and functionally distinct multiprotein complexes, termed mTORC1 and mTORC2. Both complexes contain mTOR kinase as well as the proteins mLST8 (mammalian lethal with SEC thirteen 8) (5–7), and deptor (DEP domain-containing mTOR-interacting protein) (8). mTORC1 contains the specific scaffold protein raptor (regulatory-associated protein of mTOR) (9, 10), whereas mTORC2 contains the specific binding partners rictor (rapamycin-insensitive companion of mTOR) (5–7), mSIN1 (TORC2 subunit MAPKAP1) (11–13), and PRR5/L (proline rich protein 5/-like) (14–16). The small macrolide rapamycin acutely inhibits mTORC1, but can also have long-term effects on mTORC2 (17, 18). More recently, ATP-analogs (19) that block both mTOR complexes, such as Torin 1 (20), have been developed. As rapamycin has already been available for several decades, our knowledge of signaling events associated with mTORC1 as well as its metabolic inputs and outputs is much broader as compared with mTORC2. mTORC1 responds to growth factors (insulin), nutrients (amino acids, aa) and energy (ATP). In response, mTORC1 activates anabolic processes (protein, lipid, nucleotide synthesis) and blocks catabolic processes (autophagy) to ultimately allow cellular growth (21). The insulin signal is transduced to mTORC1 via the insulin receptor (IR), and the insulin receptor substrate (IRS), which associates with class I phosphoinositide 3-kinases (PI3Ks). Subsequent phosphatidylinositol 3,4,5 trisphosphate (PIP3) binding leads to relocalization of the AGC kinases phosphoinositide-dependent protein kinase 1 (PDK1) and Akt (also termed protein kinase B, PKB) to the plasma membrane, where PDK1 phosphorylates Akt at T308 (22, 23). In response, Akt phosphorylates and inhibits the heterocomplex formed by the tuberous sclerosis complex proteins 1 and 2 (TSC1-TSC2) (24, 25). TSC1-TSC2 is the inhibitory, GTPase-activating protein for the mTORC1-inducing GTPase Ras homolog enriched in brain (rheb) (26–30), which activates mTORC1 at the lysosome. mTORC1 localization depends on the presence of aa, which in a rag GTPase-dependent manner induce mTORC1 relocalization to lysosomes (31, 32). Low energy levels are sensed by the AMP-dependent kinase (AMPK), which in turn phosphorylates the TSC1-TSC2 complex (33) and raptor (34), thereby inhibiting mTORC1.mTORC1 phosphorylates its well-described downstream substrate S6-kinase (S6K) at T389, the proline-rich Akt substrate of 40 kDa (PRAS40) at S183, and the translational repressor 4E-binding protein (4E-BP) at T37/46 (35–41). Unphosphorylated 4E-BP binds and inhibits the translation initiation factor 4G (eIF4G), which within the eIF4F complex mediates the scanning process of the ribosome to reach the start codon. Phosphorylation by mTORC1 inhibits 4E-BP''s interaction with eIF4E, thus allowing for assembly of eIF4F, and translation initiation (42, 43). More recently, also the IR-activating growth factor receptor-bound protein 10 (Grb10) (44, 45), the autophagy-initiating Unc-51-like kinase ULK1 (46), and the trifunctional enzymatic complex CAD composed of carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (47, 48), which is required for nucleotide synthesis, have been described as direct mTORC1 substrates.mTORC2 activation is mostly described to be mediated by insulin, and this is mediated by a PI3K variant that is distinct from the PI3K upstream of mTORC1 (49, 50). Furthermore, mTORC2 responds to aa (5, 51). In response, mTORC2 phosphorylates the AGC kinases Akt at S473 (52–55), and serum and glucocorticoid kinase SGK (56) and protein kinase C alpha (PKCalpha) (7) within their hydrophobic motifs (57, 58), to control cellular motility (5–7), hepatic glycolysis, and lipogenesis (59). In addition, mTOR autophosphorylation at S2481 has been established as an mTORC2 readout in several cell lines including HeLa cells (49).Given the multiplicity of effects via which mTOR controls cellular and organismal growth and metabolism, it is surprising that only relatively few direct mTOR substrates have been established to date. Proteomic studies are widely used to identify novel interactors and substrates of protein kinases. Two studies have recently shed light on the interaction of rapamycin and ATP-analog mTOR inhibitors with TSC2 inhibition in mammalian cells (44, 45), and one study has analyzed the effects of raptor and rictor knockouts in non-stimulated cells (48).In this work, we report a functional proteomics approach to study mTORC1 substrates. We used an inducible raptor knockdown to inhibit mTORC1 in HeLa cells, and analyzed the effect in combination with insulin and aa induction by quantitative phosphoproteomics using stable isotope labeling by amino acids in cell culture (SILAC) (60). In parallel, we purified endogenous mTOR complexes and studied the interactome of mTOR by SILAC-MS. Through comparative data evaluation, we identified acinus L as a potential novel aa/insulin-sensitive mTOR substrate. We further validated acinus L by co-immunoprecipitation and MS-enhanced kinase assays as a new direct mTORC1 substrate.     

Nanosized paclitaxel-loaded niosomes: formulation,in vitro cytotoxicity,and apoptosis gene expression in breast cancer cell lines

Molecular Biology Reports - In this study, the optimized niosomal formulation containing paclitaxel using non-ionic surfactants and cholesterol was designed and its cytotoxic effects against...     

Parameters Effects Evaluation of Microbial Strengthening of Sandy Soils in Mixing Experiments Using Taguchi Methodology

The effects of experimental parameters including soil type, curing duration, inoculum size, and biomass and nutrients concentration on soil strengthening due to calcite precipitation by Sporosarcina pasteurii PTCC 1645 were investigated. The laboratory-scale mixing experiments on remolded samples were designed by the Taguchi method. Soil type proved to be the most incorporating factor, followed by curing time and nutrient concentration. The main effect and the interactions of the parameters were presented and the optimal conditions were obtained. This suggests the importance of local conditions including soil type on any future large-scale, in situ application.     

Phylogenetic Analysis of Clinically Relevant Fusarium Species in Iran

Mycopathologia - Fungi of the genus Fusarium are well known as major plant pathogens but also cause a broad spectrum of human infections. Sixty-three clinical isolates, collected during...     

Characterization of Aqueous Dispersions and Gels Made of Sodium Caseinate and Basil Seed Gum: Phase Behavior,Rheology, and Microstructure

The interactions between sodium caseinate (NaCas) and basil seed gum (BSG) in the presence of calcium chloride (CaCl₂) were investigated. The phase behavior of the mixed aqueous dispersions and their gels revealed a homogeneous mixture, obtained at the higher concentrations of both CaCl₂ and BSG. The Herschel-Bulkley model sufficiently fitted the flow behavior of the mixture solution data. Apparent viscosity increased significantly (p < 0.05) by increasing the concentration of BSG, where the addition of CaCl₂ had no significant effect on the viscosity of the samples (p > 0.05). Furthermore, there was an increase in thixotropy due to the higher concentrations of BSG and CaCl₂. Based on the frequency sweep test, at the low frequencies, a more gel-like behavior was observed in the case of the higher concentrations of either BSG or CaCl₂. The rheological and SEM data suggested that the stronger structure of NaCas-BSG gel in the presence of the higher concentrations of CaCl₂ was related to the induction of complex formation between the two biopolymers.

     

Synthesis of Iron-Amino Acid Chelates and Evaluation of Their Efficacy as Iron Source and Growth Stimulator for Tomato in Nutrient Solution Culture

Supplying a sufficient amount of available iron (Fe) for plant growth in hydroponic nutrient solutions is a great challenge. The chelators commonly used to supply Fe in nutrient solutions have several disadvantages and may negatively affect plant growth. In this research study we have synthesized certain Fe-amino acid chelates, including Fe-arginine [Fe(Arg)₂], Fe-glycine [Fe(Gly)₂], and Fe-histidine [Fe(His)₂], and evaluated their efficacy as an Fe source for two tomato cultivars (Lycopersicon esculentum Mill. cvs. ‘Rani’ and ‘Sarika’) grown in nutrient solution. Application of Fe-amino acid chelates significantly increased root and shoot dry matter yield of both tomato cultivars compared with Fe-EDTA. Tomato plants supplied with Fe-amino acid chelates also accumulated significantly higher levels of Fe, Zn, and N in their roots and shoots compared with those supplied with Fe-EDTA. In ‘Sarika’, the effect of Fe-amino acid chelates on shoot Fe content was in the order Fe(His)₂?>?Fe(Gly)₂?>?Fe(Arg)₂. In ‘Rani’, the addition of all synthesized Fe-amino acid chelates significantly increased activity of ascorbate peroxidase (APX) in comparison with Fe-EDTA, whereas in ‘Sarika’, only Fe(His)₂ increased shoot APX activity. The results obtained indicated that using Fe-amino acid chelates in the nutrient solution could supply a sufficient amount of Fe for plant uptake and also improve root and shoot growth of tomato plants, although this increase was cultivar-dependent. According to the results, Fe-amino acid chelates can be used as an alternative for Fe-EDTA to supply Fe in nutrient solutions.     

Evaluation and optimization of ethanol production from carob pod extract by <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> using response surface methodology

In this research, ethanol production from carob pod extract (extract) using Zymomonas mobilis with medium optimized by Plackett–Burman (P–B) and response surface methodologies (RSM) was studied. Z. mobilis was recognized as useful for ethanol production from carob pod extract. The effects of initial concentrations of sugar, peptone, and yeast extract as well as agitation rate (rpm), pH, and culture time in nonhydrolyzed carob pod extract were investigated. Significantly affecting variables (P = 0.05) in the model obtained from RSM studies were: weights of bacterial inoculum, initial sugar, peptone, and yeast extract. Acid hydrolysis was useful to complete conversion of sugars to glucose and fructose. Nonhydrolyzed extract showed higher ethanol yield and residual sugar compared with hydrolyzed extract. Ethanol produced (g g⁻¹ initial sugar, as the response) was not significantly different (P = 0.05) when Z. mobilis performance was compared in hydrolyzed and nonhydrolyzed extract. The maximum ethanol of 0.34 ± 0.02 g g⁻¹ initial sugar was obtained at 30°C, initial pH 5.2, and 80 rpm, using concentrations (g per 50 mL culture media) of: inoculum bacterial dry weight, 0.017; initial sugar, 5.78; peptone, 0.43; yeast extract, 0.43; and culture time of 36 h.     

Bioaccumulation of Trace Elements in Trophic Levels of Wetland Plants and Waterfowl Birds

Present study investigates relationships between total and bioaccessibility of trace elements (Cd, Co, Cr, Cu, Mn, NI, Pb, V, and Zn) concentrations in sediment and their bioaccumulation in species in Shadegan wetland in southwest of Iran. Bioavailability factor (BAF) and translocation factor (TF) were calculated in plants and trophic transfer factor (TTF) was determined in bird species. For this purpose, sampling of sediments, aquatic plants including Phragmites australis, Typha australis, Scripus maritimus and two bird species encircling Porphyrio porphyrio and globally threatened Marmaronetta angustirostris were carried out during winter 2009. Result of chemical analysis show that bioaccessibility concentrations of Mn (8.31 mg/kg), V (1.33 mg/kg), and Pb (1.03 mg/kg) are higher than other metals. The uptake trend of trace elements in plant decreases as root > stem > leaf. Accumulation levels of trace elements in different tissues of P. porphyrio and M. angustirostris are almost identical and considerable. Accumulation and toxicity of Cd in birds is more than plants. In addition, BAF of V, Pb, and Cr indicates high accumulation by plants and great pollution rate in the area of study. In S. maritimus TF for Mn, Cu, Pb, and V are high whereas in T. australis, Cu and Pb posses the highest TF. Also Cr, Co, Mn, Ni, and Zn have higher TF from stem to leaf than root to stem in P. australis. Finally, TTFs were compared in various bird species.     

Genetic diversity analysis of Zingiber Officinale Roscoe by RAPD collected from subcontinent of India

The present investigation was undertaken for the assessment of 12 accessions of Zingiber officinale Rosc. collected from subcontinent of India by RAPD markers. DNA was isolated using CTAB method. Thirteen out of twenty primers screened were informative and produced 275 amplification products, among which 261 products (94.90%) were found to be polymorphic. The percentage polymorphism of all 12 accessions ranged from 88.23% to 100%. Most of the RAPD markers studied showed different levels of genetic polymorphism. The data of 275 RAPD bands were used to generate Jaccard’s similarity coefficients and to construct a dendrogram by means of UPGMA. Results showed that ginger undergoes genetic variation due to a wide range of ecological conditions. This investigation was an understanding of genetic variation within the accessions. It will also provide an important input into determining resourceful management strategies and help to breeders for ginger improvement program.