A road map for molecular ecology

The discipline of molecular ecology has undergone enormous changes since the journal bearing its name was launched approximately two decades ago. The field has seen great strides in analytical methods development, made groundbreaking discoveries and experienced a revolution in genotyping technology. Here, we provide brief perspectives on the main subdisciplines of molecular ecology, describe key questions and goals, discuss common challenges, predict future research directions and suggest research priorities for the next 20 years.     

Sunflower genetic,genomic and ecological resources

Long a major focus of genetic research and breeding, sunflowers (Helianthus) are emerging as an increasingly important experimental system for ecological and evolutionary studies. Here, we review the various attributes of wild and domesticated sunflowers that make them valuable for ecological experimentation and describe the numerous publicly available resources that have enabled rapid advances in ecological and evolutionary genetics. Resources include seed collections available from germplasm centres at the USDA and INRA, genomic and EST sequences, mapping populations, genetic markers, genetic and physical maps and other forward‐ and reverse‐genetic tools. We also discuss some of the key evolutionary, genetic and ecological questions being addressed in sunflowers, as well as gaps in our knowledge and promising areas for future research.     

Skin and Soft Tissue Infections and Associated Complications among Commercially Insured Patients Aged 0–64 Years with and without Diabetes in the U.S.

Introduction

Skin and soft tissue infections (SSTIs) are common infections occurring in ambulatory and inpatient settings. The extent of complications associated with these infections by diabetes status is not well established.

Methods

Using a very large repository database, we examined medical and pharmacy claims of individuals aged 0–64 between 2005 and 2010 enrolled in U.S. health plans. Diabetes, SSTIs, and SSTI-associated complications were identified by ICD-9 codes. SSTIs were stratified by clinical category and setting of initial diagnosis.

Results

We identified 2,227,401 SSTI episodes, 10% of which occurred in diabetic individuals. Most SSTIs were initially diagnosed in ambulatory settings independent from diabetes status. Abscess/cellulitis was the more common SSTI group in diabetic and non-diabetic individuals (66% and 59%, respectively). There were differences in the frequencies of SSTI categories between diabetic and non-diabetic individuals (p<0.01). Among SSTIs diagnosed in ambulatory settings, the SSTI-associated complication rate was over five times higher in people with diabetes than in people without diabetes (4.9% vs. 0.8%, p<0.01) and SSTI-associated hospitalizations were 4.9% and 1.1% in patients with and without diabetes, respectively. Among SSTIs diagnosed in the inpatient setting, bacteremia/endocarditis/septicemia/sepsis was the most common associated complication occurring in 25% and 16% of SSTIs in patients with and without diabetes, respectively (p<0.01).

Conclusions

Among persons with SSTIs, we found SSTI-associated complications were five times higher and SSTI-associated hospitalizations were four times higher, in patients with diabetes compared to those without diabetes. SSTI prevention efforts in individuals with diabetes may have significant impact on morbidity and healthcare resource utilization.     

Massive excretion of calcium oxalate from late prepupal salivary glands of Drosophila melanogaster demonstrates active nephridial‐like anion transport

The Drosophila salivary glands (SGs) were well known for the puffing patterns of their polytene chromosomes and so became a tissue of choice to study sequential gene activation by the steroid hormone ecdysone. One well‐documented function of these glands is to produce a secretory glue, which is released during pupariation to fix the freshly formed puparia to the substrate. Over the past two decades SGs have been used to address specific aspects of developmentally‐regulated programmed cell death (PCD) as it was thought that they are doomed for histolysis and after pupariation are just awaiting their fate. More recently, however, we have shown that for the first 3–4 h after pupariation SGs undergo tremendous endocytosis and vacuolation followed by vacuole neutralization and membrane consolidation. Furthermore, from 8 to 10 h after puparium formation (APF) SGs display massive apocrine secretion of a diverse set of cellular proteins. Here, we show that during the period from 11 to 12 h APF, the prepupal glands are very active in calcium oxalate (CaOx) extrusion that resembles renal or nephridial excretory activity. We provide genetic evidence that Prestin, a Drosophila homologue of the mammalian electrogenic anion exchange carrier SLC26A5, is responsible for the instantaneous production of CaOx by the late prepupal SGs. Its positive regulation by the protein kinases encoded by fray and wnk lead to increased production of CaOx. The formation of CaOx appears to be dependent on the cooperation between Prestin and the vATPase complex as treatment with bafilomycin A₁ or concanamycin A abolishes the production of detectable CaOx. These data demonstrate that prepupal SGs remain fully viable, physiologically active and engaged in various cellular activities at least until early pupal period, that is, until moments prior to the execution of PCD.     

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.     

Mouse models of the laminopathies

The A and B type lamins are nuclear intermediate filament proteins that comprise the bulk of the nuclear lamina, a thin proteinaceous structure underlying the inner nuclear membrane. The A type lamins are encoded by the lamin A gene (LMNA). Mutations in this gene have been linked to at least nine diseases, including the progeroid diseases Hutchinson-Gilford progeria and atypical Werner's syndromes, striated muscle diseases including muscular dystrophies and dilated cardiomyopathies, lipodystrophies affecting adipose tissue deposition, diseases affecting skeletal development, and a peripheral neuropathy. To understand how different diseases arise from different mutations in the same gene, mouse lines carrying some of the same mutations found in the human diseases have been established. We, and others have generated mice with different mutations that result in progeria, muscular dystrophy, and dilated cardiomyopathy. To further our understanding of the functions of the lamins, we also created mice lacking lamin B1, as well as mice expressing only one of the A type lamins. These mouse lines are providing insights into the functions of the lamina and how changes to the lamina affect the mechanical integrity of the nucleus as well as signaling pathways that, when disrupted, may contribute to the disease.