Morphological Diversity of the Rod Spherule: A Study of Serially Reconstructed Electron Micrographs

Purpose

Rod spherules are the site of the first synaptic contact in the retina’s rod pathway, linking rods to horizontal and bipolar cells. Rod spherules have been described and characterized through electron micrograph (EM) and other studies, but their morphological diversity related to retinal circuitry and their intracellular structures have not been quantified. Most rod spherules are connected to their soma by an axon, but spherules of rods on the surface of the Mus musculus outer plexiform layer often lack an axon and have a spherule structure that is morphologically distinct from rod spherules connected to their soma by an axon. Retraction of the rod axon and spherule is often observed in disease processes and aging, and the retracted rod spherule superficially resembles rod spherules lacking an axon. We hypothesized that retracted spherules take on an axonless spherule morphology, which may be easier to maintain in a diseased state. To test our hypothesis, we quantified the spatial organization and subcellular structures of rod spherules with and without axons. We then compared them to the retracted spherules in a disease model, mice that overexpress Dscam (Down syndrome cell adhesion molecule), to gain a better understanding of the rod synapse in health and disease.

Methods

We reconstructed serial EM images of wild type and Dscam^GoF (gain of function) rod spherules at a resolution of 7 nm in the X-Y axis and 60 nm in the Z axis. Rod spherules with and without axons, and retracted spherules in the Dscam^GoF retina, were reconstructed. The rod spherule intracellular organelles, the invaginating dendrites of rod bipolar cells and horizontal cell axon tips were also reconstructed for statistical analysis.

Results

Stereotypical rod (R1) spherules occupy the outer two-thirds of the outer plexiform layer (OPL), where they present as spherical terminals with large mitochondria. This spherule group is highly uniform and composed more than 90% of the rod spherule population. Rod spherules lacking an axon (R2) were also described and characterized. This rod spherule group consists of a specific spatial organization that is strictly located at the apical OPL-facing layer of the Outer Nuclear Layer (ONL). The R2 spherule displays a large bowl-shaped synaptic terminal that hugs the rod soma. Retracted spherules in the Dscam^GoF retina were also reconstructed to test if they are structurally similar to R2 spherules. The misplaced rod spherules in Dscam^GoF have a gross morphology that is similar to R2 spherules but have significant disruption in internal synapse organization.

Conclusion

We described a morphological diversity within Mus musculus rod spherules. This diversity is correlated with rod location in the ONL and contributes to the intracellular differences within spherules. Analysis of the Dscam^GoF retina indicated that their R2 spherules are not significantly different than wild type R2 spherules, but that their retracted rod spherules have abnormal synaptic organization.     

Heterologous Expression Screens in Nicotiana benthamiana Identify a Candidate Effector of the Wheat Yellow Rust Pathogen that Associates with Processing Bodies

Rust fungal pathogens of wheat (Triticum spp.) affect crop yields worldwide. The molecular mechanisms underlying the virulence of these pathogens remain elusive, due to the limited availability of suitable molecular genetic research tools. Notably, the inability to perform high-throughput analyses of candidate virulence proteins (also known as effectors) impairs progress. We previously established a pipeline for the fast-forward screens of rust fungal candidate effectors in the model plant Nicotiana benthamiana. This pipeline involves selecting candidate effectors in silico and performing cell biology and protein-protein interaction assays in planta to gain insight into the putative functions of candidate effectors. In this study, we used this pipeline to identify and characterize sixteen candidate effectors from the wheat yellow rust fungal pathogen Puccinia striiformis f sp tritici. Nine candidate effectors targeted a specific plant subcellular compartment or protein complex, providing valuable information on their putative functions in plant cells. One candidate effector, {"type":"entrez-protein","attrs":{"text":"PST02549","term_id":"1371656274"}}PST02549, accumulated in processing bodies (P-bodies), protein complexes involved in mRNA decapping, degradation, and storage. {"type":"entrez-protein","attrs":{"text":"PST02549","term_id":"1371656274"}}PST02549 also associates with the P-body-resident ENHANCER OF mRNA DECAPPING PROTEIN 4 (EDC4) from N. benthamiana and wheat. We propose that P-bodies are a novel plant cell compartment targeted by pathogen effectors.     

A Unique Combination of Nutritionally Active Ingredients Can Prevent Several Key Processes Associated with Atherosclerosis In Vitro

Introduction

Atherosclerosis is the underlying cause of cardiovascular disease that leads to more global mortalities each year than any other ailment. Consumption of active food ingredients such as phytosterols, omega-3 polyunsaturated fatty acids and flavanols are known to impart beneficial effects on cardiovascular disease although the combined actions of such agents in atherosclerosis is poorly understood. The aim of this study was to screen a nutritional supplement containing each of these active components for its anti-atherosclerotic effect on macrophages in vitro.

Results

The supplement attenuated the expression of intercellular adhesion molecule-1 and macrophage chemoattractant protein-1 in human and murine macrophages at physiologically relevant doses. The migratory capacity of human monocytes was also hindered, possibly mediated by eicosapentaenoic acid and catechin, while the ability of foam cells to efflux cholesterol was improved. The polarisation of murine macrophages towards a pro-inflammatory phenotype was also attenuated by the supplement.

Conclusion

The formulation was able to hinder multiple key steps of atherosclerosis development in vitro by inhibiting monocyte recruitment, foam cell formation and macrophage polarisation towards an inflammatory phenotype. This is the first time a combination these ingredients has been shown to elicit such effects and supports its further study in preclinical in vivo models.     

GAP promoter‐based fed‐batch production of highly bioactive core streptavidin by Pichia pastoris

Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin‐auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol‐based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost. Moreover, common methanol‐based processes lead to large proportions of biotin‐blocked binding sites of streptavidin due to biotin‐supplemented media. Targeting these problems, this paper provides strategies for the methanol‐free production of highly bioactive core streptavidin by P. pastoris under control of the constitutive GAP promoter. Complex were superior to synthetic production media regarding the proportion of biotin‐blocked streptavidin. The optimized, easily scalable fed‐batch process led to a tetrameric product concentration of up to 4.16 ± 0.11 µM of biotin‐free streptavidin and a productivity of 57.8 nM h^?1 based on constant glucose feeding and a successive shift of temperature and pH throughout the cultivation, surpassing the concentration in un‐optimized conditions by a factor of 3.4. Parameter estimation indicates that the optimized conditions caused a strongly increased accumulation of product at diminishing specific growth rates (μ ≈ D < 0.01 h^?1), supporting the strategy of feeding. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:855–864, 2016     

Engineering Triterpene and Methylated Triterpene Production in Plants Provides Biochemical and Physiological Insights into Terpene Metabolism

Linear, branch-chained triterpenes, including squalene (C30), botryococcene (C30), and their methylated derivatives (C31–C37), generated by the green alga Botryococcus braunii race B have received significant attention because of their utility as chemical and biofuel feedstocks. However, the slow growth habit of B. braunii makes it impractical as a production system. In this study, we evaluated the potential of generating high levels of botryococcene in tobacco (Nicotiana tabacum) plants by diverting carbon flux from the cytosolic mevalonate pathway or the plastidic methylerythritol phosphate pathway by the targeted overexpression of an avian farnesyl diphosphate synthase along with two versions of botryococcene synthases. Up to 544 µg g⁻¹ fresh weight of botryococcene was achieved when this metabolism was directed to the chloroplasts, which is approximately 90 times greater than that accumulating in plants engineered for cytosolic production. To test if methylated triterpenes could be produced in tobacco, we also engineered triterpene methyltransferases (TMTs) from B. braunii into wild-type plants and transgenic lines selected for high-level triterpene accumulation. Up to 91% of the total triterpene contents could be converted to methylated forms (C31 and C32) by cotargeting the TMTs and triterpene biosynthesis to the chloroplasts, whereas only 4% to 14% of total triterpenes were methylated when this metabolism was directed to the cytoplasm. When the TMTs were overexpressed in the cytoplasm of wild-type plants, up to 72% of the total squalene was methylated, and total triterpene (C30+C31+C32) content was elevated 7-fold. Altogether, these results point to innate mechanisms controlling metabolite fluxes, including a homeostatic role for squalene.Terpenes and terpenoids represent a distinct class of natural products (Buckingham, 2003) that are derived from two universal five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In eukaryotic fungi and animals, IPP and DMAPP are synthesized via the mevalonate (MVA) pathway, whereas in prokaryotes, they are synthesized via the methylerythritol phosphate (MEP) pathway. In higher plants, the pathways are present in separate compartments and are believed to operate independently. The MVA pathway in the cytoplasm is predominantly responsible for sesquiterpene (C15), triterpene (C30), and polyprenol (greater than C45) biosynthesis and associated with the endoplasmic reticulum (ER) system. The MEP pathway resides in plastids and is dedicated to monoterpenes (C10), diterpenes (C20), carotenoids (C40), and long-chain phytol biosynthesis. All these compounds are usually produced by plants for a variety of physiological (i.e. hormones, aliphatic membrane anchors, and maintaining membrane structure) and ecological (i.e. defense compounds and insect/animal attractants) roles (Kempinski et al., 2015). Terpenes are also important for various industrial applications, ranging from flavors and fragrances (Schwab et al., 2008) to medicines (Dewick, 2009; Niehaus et al., 2011; Shelar, 2011).The utility of terpenes as chemical and biofuel feedstocks has also received considerable attention recently. Isoprenoid-derived biofuels include farnesane (Renninger and McPhee, 2008; Rude and Schirmer, 2009), bisabolene (Peralta-Yahya et al., 2011), pinene dimers (Harvey et al., 2010), isopentenal (Withers et al., 2007), and botryococcene (Moldowan and Seifert, 1980; Hillen et al., 1982; Glikson et al., 1989; Mastalerz and Hower, 1996). The richness of branches within these hydrocarbon scaffolds correlate with their high-energy content, which enables them to serve as suitable alternatives to crude petroleum (Peralta-Yahya and Keasling, 2010). Indeed, some of them are already major contributors to current-day petroleum-based fuels. One of the best examples of this is the triterpene oil accumulating in the green alga Botryococcus braunii race B, which is considered a major progenitor to oil and coal shale deposits (Moldowan and Seifert, 1980). This alga has been well studied, and the major constituents of its prodigious hydrocarbon oil are a group of triterpenes including squalene (C30), organism-specific botryococcene (C30), methylated squalene (C31–C34), and methylated botryococcene (C31–C37; Metzger et al., 1988; Huang and Poulter, 1989; Okada et al., 1995), which can be readily converted into all classes of combustible fuels under hydrocracking conditions (Hillen et al., 1982).The unique biosynthetic mechanism for the triterpenes in B. braunii was recently described by Niehaus et al. (2011), and a series of novel squalene synthase-like genes were identified (Fig. 1). In short, squalene synthase-like enzyme, SSL-1, performs a head-to-head condensation of two farnesyl diphosphate (FPP) molecules into presqualene diphosphate, followed by a reductive rearrangement to yield squalene (C30) by the enzyme SSL-2, or is converted by SSL-3 to form botryococcene through a different reductive rearrangement (Niehaus et al., 2011). Methylated derivatives are the dominant triterpene species generated by B. braunii race B (Metzger, 1985; Metzger et al., 1988), and these derivatives are known to yield higher quality fuels due to their high energy content and the hydrocracking products derived by virtue of having more hydrocarbon branches. Triterpene methyltransferases (TMTs) that can methylate squalene and botryococcene have been successfully characterized by Niehaus et al. (2012). TRITERPENE METHYLTRANSFERASE1 (TMT-1) and TMT-2 prefer squalene C30 as their substrate for the production of monomethylated (C31) or dimethylated (C32) squalene, while TMT-3 prefers botryococcene as its substrate for the biosynthesis of monomethylated (C31) or dimethylated (C32) botryococcene (Fig. 1). These TMTs are believed to be insoluble enzymes; they exhibit large hydrophobic areas, and their activities were only observed in vitro using yeast microsomal preparations (no activity was observed when expressed in bacteria; Niehaus et al., 2012).Open in a separate windowFigure 1.Depiction of the catalytic roles of novel SSL and TMT enzymes in B. braunii race B and their putative contributions to the triterpene constituents (Niehaus et al., 2011; Niehaus et al., 2012). SSL-1 catalyzes the condensation of two farnesyl diphosphate (FPP) molecules to presqualene diphosphate (PSPP), which is converted to either squalene or botryococcene by SSL-2 or SSL-3, respectively. Squalene can also be synthesized directly from the condensation of two FPP molecules catalyzed by squalene synthase (SQS). TMT-1 and TMT-2 transfer the methyl donor group from S-adenosylmethionine (SAM) to squalene to form monomethylated and dimethylated squalene, whereas TMT-3 acts on botryococcene to form monomethylated and dimethylated botryococcene (Niehaus et al., 2012).Like the majority of identified methyltransferases, these TMTs utilize the methyl donor S-adenosyl methionine (SAM), which is ubiquitous in prokaryotes and eukaryotes (Scheer et al., 2011; Liscombe et al., 2012). In plants, SAM is one of the most abundant cofactors (Fontecave et al., 2004; Sauter et al., 2013) and is synthesized exclusively in the cytosol (Wallsgrove et al., 1983; Ravanel et al., 1998, 2004; Bouvier et al., 2006). While it is used predominantly as a methyl donor in the methylation reaction (Ravanel et al., 2004), it also serves as the primary precursor for the biosynthesis of ethylene (Wang et al., 2002b), polyamines (Kusano et al., 2008), and nicotianamine (Takahashi et al., 2003), which play a variety of important roles for plant growth and development (Huang et al., 2012; Sauter et al., 2013). The SAM present in organelles, like the chloroplast, appears to be imported from the cytosol by specific SAM/S-adenosylhomocysteine exchange transporters that reside on the envelope membranes of plastids (Ravanel et al., 2004; Bouvier et al., 2006). The imported SAM is involved in the biogenesis of Asp-derived amino acids (Curien et al., 1998; Jander and Joshi, 2009; Sauter et al., 2013) and serves as the methyl donor for the methylation of macromolecules, such as plastid DNA (Nishiyama et al., 2002; Ahlert et al., 2009) and proteins (Houtz et al., 1989; Niemi et al., 1990; Ying et al., 1999; Trievel et al., 2003; Alban et al., 2014), and small molecule metabolites, such as prenylipids (e.g. plastoquinone, tocopherol, chlorophylls, and phylloquinone; Bouvier et al., 2005, 2006; DellaPenna, 2005).Although plants and microbes are the natural sources for useful terpenes, most of them are produced in very small amounts and often as complex mixtures. In contrast, B. braunii produces large quantities of triterpenes, but its slow growth makes it undesirable as a viable production platform (Niehaus et al., 2011). Nevertheless, metabolic engineering and synthetic biology offer many strategies to manipulate terpene metabolism in various biological systems to achieve high-value terpene production with high yield and high fidelity for particular practical applications (Nielsen and Keasling, 2011). Many successes have been achieved in engineering valuable terpenes in heterotrophic microbes, such as Escherichia coli (Nishiyama et al., 2002; Martin et al., 2003; Ajikumar et al., 2010) and Saccharomyces cerevisiae (Ro et al., 2006; Takahashi et al., 2007; Westfall et al., 2012; Zhuang and Chappell, 2015). The strategies developed in these efforts usually take advantage of specific microbe strains whose innate biosynthetic machinery is genetically modified to accumulate certain prenyldiphosphate precursors (e.g. IPP or FPP), which can be utilized by other introduced terpene synthase(s) for the production of the desired terpene(s). For example, greater than 900 mg L⁻¹ bisabolene was produced when bisabolene synthase genes from plants were introduced into FPP-overproducing E. coli or S. cerevisiae strains (Peralta-Yahya et al., 2011). High levels of farnesane production for diesel fuels were also achieved by reductive hydrogenation of its precursor farnesene, which was generated from a genetically engineered yeast (e.g. Saccharomyces cerevisiae) strain using plant farnesene synthases (Renninger and McPhee, 2008; Ubersax and Platt, 2010). However, terpene production using microbial platforms is still dependent on exogenous feedstocks (i.e. sugars) and elaborate production facilities, both of which add significantly to their production costs.Compared with microbial systems, engineering terpene production in plant systems seems like an attractive target as well. This is because plants can take advantage of photosynthesis by using atmospheric CO₂ as their carbon resource instead of relying on exogenous carbon feedstocks. Moreover, crop plants such as tobacco (Nicotiana tabacum) can generate a large amount of green tissues efficiently when grown for biomass production (Schillberg et al., 2003; Andrianov et al., 2010), making them a robust, sustainable, and scalable platform for large-scale terpene production. Nonetheless, compared with microbial platforms, there are only a few examples of elevating terpene production in bioengineered plants. This is due partly to higher plants being complex multicellular organisms, in which terpene metabolism generally utilizes more complex innate machinery that can be compartmentalized intracellularly and to cell/tissue specificities (Lange and Ahkami, 2013; Kempinski et al., 2015). Significant efforts have been made to overcome these obstacles to improve the production of valuable terpenes in plants, including monoterpenes (Lücker et al., 2004; Ohara et al., 2010; Lange et al., 2011), sesquiterpenes (Aharoni et al., 2003; Kappers et al., 2005; Wu et al., 2006; Davidovich-Rikanati et al., 2008), diterpenes (Besumbes et al., 2004; Anterola et al., 2009), and triterpenes (Inagaki et al., 2011; Wu et al., 2012). Among these, engineering terpene metabolism into a subcellular organelle, where the engineered enzymes/pathways can utilize unlimited/unregulated precursors as substrates, appears most successful. For example, Wu et al. (2006, 2012) expressed an avian farnesyl diphosphate synthase (FPS) with foreign sesquiterpene/triterpene synthases targeted to the plastid to divert the IPP/DMAPP pool from the plastidic MEP pathway to synthesize high levels of the novel sesquiterpenes patchoulol and amorpha-4,11-diene up to 30 µg g⁻¹ fresh weight and the triterpene squalene up to 1,000 µg g⁻¹ fresh weight. This strategy appears to be particularly robust because it avoids possible endogenous regulation of sesquiterpene and triterpene biosynthesis, which occurs normally in the cytoplasm, and relies upon more plastic precursor pools of IPP/DMAPP inherent in the plastid, which are primarily derived from the local CO₂ fixation (Wright et al., 2014).The goal of this study was to evaluate the prospects for engineering advanced features of triterpene metabolism from B. braunii into tobacco and, thus, to probe the innate intricacies of isoprenoid metabolism in plants. In order to achieve this, we first introduced the key steps of botryococcene biosynthesis into specific subcellular compartments of tobacco cells under the direction of constitutive or trichome-specific promoters. The transgenic lines expressing the enzymes in the chloroplast were found to accumulate the highest levels of botryococcene. Triterpene methyltransferases were next introduced into the same intracellular compartments of selected high-triterpene-accumulating lines. A high yield of methylated triterpenes was also achieved in transgenic lines when the TMTs were targeted to the chloroplast. Through careful comparison of the levels of triterpenes and the methylated triterpene products in the various transgenic lines, we have also gained a deeper insight into the subcellular distribution of the triterpene products in these transgenic lines as well as a better understanding of methylation metabolism for specialized metabolites in particular compartments. These findings all contribute to our understanding of the regulatory elements that control carbon flux through the innate terpene biosynthetic pathways operating in plants.     

Rust fungal effectors mimic host transit peptides to translocate into chloroplasts

Parasite effector proteins target various host cell compartments to alter host processes and promote infection. How effectors cross membrane‐rich interfaces to reach these compartments is a major question in effector biology. Growing evidence suggests that effectors use molecular mimicry to subvert host cell machinery for protein sorting. We recently identified chloroplast‐targeted protein 1 (CTP1), a candidate effector from the poplar leaf rust fungus Melampsora larici‐populina that carries a predicted transit peptide and accumulates in chloroplasts and mitochondria. Here, we show that the CTP1 transit peptide is necessary and sufficient for accumulation in the stroma of chloroplasts. CTP1 is part of a Melampsora‐specific family of polymorphic secreted proteins. Two members of that family, CTP2 and CTP3, also translocate in chloroplasts in an N‐terminal signal‐dependent manner. CTP1, CTP2 and CTP3 are cleaved when they accumulate in chloroplasts, while they remain intact when they do not translocate into chloroplasts. Our findings reveal that fungi have evolved effector proteins that mimic plant‐specific sorting signals to traffic within plant cells.     

Longer lifespan in male mice treated with a weakly estrogenic agonist,an antioxidant,an α‐glucosidase inhibitor or a Nrf2‐inducer

The National Institute on Aging Interventions Testing Program (ITP) evaluates agents hypothesized to increase healthy lifespan in genetically heterogeneous mice. Each compound is tested in parallel at three sites, and all results are published. We report the effects of lifelong treatment of mice with four agents not previously tested: Protandim, fish oil, ursodeoxycholic acid (UDCA) and metformin – the latter with and without rapamycin, and two drugs previously examined: 17‐α‐estradiol and nordihydroguaiaretic acid (NDGA), at doses greater and less than used previously. 17‐α‐estradiol at a threefold higher dose robustly extended both median and maximal lifespan, but still only in males. The male‐specific extension of median lifespan by NDGA was replicated at the original dose, and using doses threefold lower and higher. The effects of NDGA were dose dependent and male specific but without an effect on maximal lifespan. Protandim, a mixture of botanical extracts that activate Nrf2, extended median lifespan in males only. Metformin alone, at a dose of 0.1% in the diet, did not significantly extend lifespan. Metformin (0.1%) combined with rapamycin (14 ppm) robustly extended lifespan, suggestive of an added benefit, based on historical comparison with earlier studies of rapamycin given alone. The α‐glucosidase inhibitor, acarbose, at a concentration previously tested (1000 ppm), significantly increased median longevity in males and 90th percentile lifespan in both sexes, even when treatment was started at 16 months. Neither fish oil nor UDCA extended lifespan. These results underscore the reproducibility of ITP longevity studies and illustrate the importance of identifying optimal doses in lifespan studies.     

Identification of Polymorphisms in the Rabbit Growth Hormone Receptor (GHR) Gene and Association with Finishing Weight in a Commercial Meat Rabbit Line

A shortcut to identify DNA markers associated with economic traits is to use a candidate gene approach that is still useful in livestock species in which molecular tools and resources are not advanced or not well developed. Mutations in the growth hormone receptor (GHR) gene associated with production traits have been already described in several livestock species. For this reason GHR could be an interesting candidate gene in the rabbit. In this study we re-sequenced all exons and non-coding regions of the rabbit GHR gene in a panel of 10 different rabbits and identified 10 single nucleotide polymorphisms (SNPs). One of them (g.63453192C>G or c.106C>G), located in exon 3 was a missense mutation (p.L36V) substituting an amino acid in a highly conserved position across all mammals. This mutation was genotyped in 297 performance tested rabbits of a meat male line and association analysis showed that the investigated SNP was associated with weight at 70 days (P < 0.05). The most frequent genotype (GG) was in animals with higher weight at this age, suggesting that the high directional selection pressure toward this trait since the constitution of the genotyped line might have contributed to shape allele frequencies at this polymorphic site.