Solasodine production from cell culture of <Emphasis Type="Italic">Solanum hainanense</Emphasis> Hance

Stem explants of Solanum hainanense Hance plantlets were cultured on Murashige and Skoog solid medium, containing 3% (w/v) sucrose, supplemented with 0.1 mg/L benzylaminopurine (BAP) and 1.0 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) for callus production. To establish the cell suspension culture, 3 g of fresh callus were cultured in 50 mL of the same medium, but without a solid agent, at an agitation speed of 120 rpm. Every 15 mL of culture was sub-cultured in fresh MS liquid medium for maintenance. The cell biomass of S. hainanense reached a maximum value of 18.47 g after 4 weeks of culture on the same MS medium, but with the sucrose content increased to 4%, at an agitation speed of 150 rpm, with 20 mL of inoculum. Analysis via high performance liquid chromatography (HPLC) showed that the solasodine content in the cell suspension after 4-weeks old (121.01 mg/g) was higher than that of in planta 1-year old roots (20.52 mg/g) by approximately 6-fold.     

Chalcone synthase and its functions in plant resistance

Chalcone synthase (CHS, EC 2.3.1.74) is a key enzyme of the flavonoid/isoflavonoid biosynthesis pathway. Besides being part of the plant developmental program the CHS gene expression is induced in plants under stress conditions such as UV light, bacterial or fungal infection. CHS expression causes accumulation of flavonoid and isoflavonoid phytoalexins and is involved in the salicylic acid defense pathway. This review will discuss CHS and its function in plant resistance.     

Combining antigen-based therapy with GABA treatment synergistically prolongs survival of transplanted ß-cells in diabetic NOD mice

Antigen-based therapies (ABTs) very effectively prevent the development of type 1 diabetes (T1D) when given to young nonobese diabetic (NOD) mice, however, they have little or no ability to reverse hyperglycemia in newly diabetic NOD mice. More importantly, ABTs have not yet demonstrated an ability to effectively preserve residual ?-cells in individuals newly diagnosed with type 1 diabetes (T1D). Accordingly, there is great interest in identifying new treatments that can be combined with ABTs to safely protect ?-cells in diabetic animals. The activation of γ-aminobutyric acid (GABA) receptors (GABA-Rs) on immune cells has been shown to prevent T1D, experimental autoimmune encephalomyelitis (EAE) and rheumatoid arthritis in mouse models. Based on GABA's ability to inhibit different autoimmune diseases and its safety profile, we tested whether the combination of ABT with GABA treatment could prolong the survival of transplanted ?-cells in newly diabetic NOD mice. Newly diabetic NOD mice were untreated, or given GAD/alum (20 or 100 μg) and placed on plain drinking water, or water containing GABA (2 or 6 mg/ml). Twenty-eight days later, they received syngenic pancreas grafts and were monitored for the recurrence of hyperglycemia. Hyperglycemia reoccurred in the recipients given plain water, GAD monotherapy, GABA monotherapy, GAD (20 μg)+GABA (2 mg/ml), GAD (20 μg)+GABA (6 mg/ml) and GAD (100 μg)+GABA (6 mg/ml) about 1, 2-3, 3, 2-3, 3-8 and 10-11 weeks post-transplantation, respectively. Thus, combined GABA and ABT treatment had a synergistic effect in a dose-dependent fashion. These findings suggest that co-treatment with GABA (or other GABA-R agonists) may provide a new strategy to safely enhance the efficacy of other therapeutics designed to prevent or reverse T1D, as well as other T cell-mediated autoimmune diseases.     

Transmigration of melanoma cells through the blood-brain barrier: role of endothelial tight junctions and melanoma-released serine proteases

Malignant melanoma represents the third common cause of brain metastasis, having the highest propensity to metastasize to the brain of all primary neoplasms in adults. Since the central nervous system lacks a lymphatic system, the only possibility for melanoma cells to reach the brain is via the blood stream and the blood-brain barrier. Despite the great clinical importance, mechanisms of transmigration of melanoma cells through the blood-brain barrier are incompletely understood. In order to investigate this question we have used an in vitro experimental setup based on the culture of cerebral endothelial cells (CECs) and the A2058 and B16/F10 melanoma cell lines, respectively. Melanoma cells were able to adhere to confluent brain endothelial cells, a process followed by elimination of protrusions and transmigration from the luminal to the basolateral side of the endothelial monolayers. The transmigration process of certain cells was accelerated when they were able to use the routes preformed by previously transmigrated melanoma cells. After migrating through the endothelial monolayer several melanoma cells continued their movement beneath the endothelial cell layer. Melanoma cells coming in contact with brain endothelial cells disrupted the tight and adherens junctions of CECs and used (at least partially) the paracellular transmigration pathway. During this process melanoma cells produced and released large amounts of proteolytic enzymes, mainly gelatinolytic serine proteases, including seprase. The serine protease inhibitor Pefabloc® was able to decrease to 44–55% the number of melanoma cells migrating through CECs. Our results suggest that release of serine proteases by melanoma cells and disintegration of the interendothelial junctional complex are main steps in the formation of brain metastases in malignant melanoma.     

A comparison of the sensitivity and fecal egg counts of the McMaster egg counting and Kato-Katz thick smear methods for soil-transmitted helminths

Background

The Kato-Katz thick smear (Kato-Katz) is the diagnostic method recommended for monitoring large-scale treatment programs implemented for the control of soil-transmitted helminths (STH) in public health, yet it is difficult to standardize. A promising alternative is the McMaster egg counting method (McMaster), commonly used in veterinary parasitology, but rarely so for the detection of STH in human stool.

Methodology/Principal Findings

The Kato-Katz and McMaster methods were compared for the detection of STH in 1,543 subjects resident in five countries across Africa, Asia and South America. The consistency of the performance of both methods in different trials, the validity of the fixed multiplication factor employed in the Kato-Katz method and the accuracy of these methods for estimating ‘true’ drug efficacies were assessed. The Kato-Katz method detected significantly more Ascaris lumbricoides infections (88.1% vs. 75.6%, p<0.001), whereas the difference in sensitivity between the two methods was non-significant for hookworm (78.3% vs. 72.4%) and Trichuris trichiura (82.6% vs. 80.3%). The sensitivity of the methods varied significantly across trials and magnitude of fecal egg counts (FEC). Quantitative comparison revealed a significant correlation (Rs >0.32) in FEC between both methods, and indicated no significant difference in FEC, except for A. lumbricoides, where the Kato-Katz resulted in significantly higher FEC (14,197 eggs per gram of stool (EPG) vs. 5,982 EPG). For the Kato-Katz, the fixed multiplication factor resulted in significantly higher FEC than the multiplication factor adjusted for mass of feces examined for A. lumbricoides (16,538 EPG vs. 15,396 EPG) and T. trichiura (1,490 EPG vs. 1,363 EPG), but not for hookworm. The McMaster provided more accurate efficacy results (absolute difference to ‘true’ drug efficacy: 1.7% vs. 4.5%).

Conclusions/Significance

The McMaster is an alternative method for monitoring large-scale treatment programs. It is a robust (accurate multiplication factor) and accurate (reliable efficacy results) method, which can be easily standardized.     

Dextran fractional clearance studies in acute dengue infection

Background

Although increased capillary permeability is the major clinical feature associated with severe dengue infections the mechanisms underlying this phenomenon remain unclear. Dextran clearance methodology has been used to investigate the molecular sieving properties of the microvasculature in clinical situations associated with altered permeability, including during pregnancy and in various renal disorders. In order to better understand the characteristics of the vascular leak associated with dengue we undertook formal dextran clearance studies in Vietnamese dengue patients and healthy volunteers.

Methodology/Principal Findings

We carried out serial clearance studies in 15 young adult males with acute dengue and evidence of vascular leakage a) during the phase of maximal leakage and b) one and three months later, as well as in 16 healthy control subjects. Interestingly we found no difference in the clearance profiles of neutral dextran solutions among the dengue patients at any time-point or in comparison to the healthy volunteers.

Conclusions/Significance

The surface glycocalyx layer, a fibre-matrix of proteoglycans, glycosaminoglycans, and plasma proteins, forms a complex with the underlying endothelial cells to regulate plasma volume within circumscribed limits. It is likely that during dengue infections loss of plasma proteins from this layer alters the permeability characteristics of the complex; physical and/or electrostatic interactions between the dextran molecules and the glycocalyx structure may temporarily restore normal function, rendering the technique unsuitable for assessing permeability in these patients. The implications for resuscitation of patients with dengue shock syndrome (DSS) are potentially important. It is possible that continuous low-dose infusions of dextran may help to stabilize the permeability barrier in patients with profound or refractory shock, reducing the need for repeated boluses, limiting the total colloid volume required. Formal clinical studies should help to assess this strategy as an alternative to conventional fluid resuscitation for severe DSS.     

A survey of new temperature-sensitive, embryonic-lethal mutations in C. elegans: 24 alleles of thirteen genes

To study essential maternal gene requirements in the early C. elegans embryo, we have screened for temperature-sensitive, embryonic lethal mutations in an effort to bypass essential zygotic requirements for such genes during larval and adult germline development. With conditional alleles, multiple essential requirements can be examined by shifting at different times from the permissive temperature of 15°C to the restrictive temperature of 26°C. Here we describe 24 conditional mutations that affect 13 different loci and report the identity of the gene mutations responsible for the conditional lethality in 22 of the mutants. All but four are mis-sense mutations, with two mutations affecting splice sites, another creating an in-frame deletion, and one creating a premature stop codon. Almost all of the mis-sense mutations affect residues conserved in orthologs, and thus may be useful for engineering conditional mutations in other organisms. We find that 62% of the mutants display additional phenotypes when shifted to the restrictive temperature as L1 larvae, in addition to causing embryonic lethality after L4 upshifts. Remarkably, we also found that 13 out of the 24 mutations appear to be fast-acting, making them particularly useful for careful dissection of multiple essential requirements. Our findings highlight the value of C. elegans for identifying useful temperature-sensitive mutations in essential genes, and provide new insights into the requirements for some of the affected loci.     

Carbon sequestration and soil fertility of tropical tree plantations and secondary forest established on degraded land

Purpose

Much tropical land requires rehabilitation but the capacity of reforestation with plantations or naturally regenerating secondary forests for overcoming soil degradation remains unclear. We hypothesised that desirable effects, including improved soil fertility and carbon sequestration, are achieved to a greater extent in Acacia mangium plantations and secondary forests than in Eucalyptus urophylla plantations.

Methods

We tested our hypothesis across soil and climate gradients in Vietnam with linear mixed-effect models and other, comparing A. mangium and E. urophylla plantations, secondary forests and pasture.

Results

A. mangium plantations and secondary forests showed a positive correlation between biomass production and desirable soils properties including increased soil carbon, nitrogen and phosphorus, and reduced bulk density. All plantations, but not secondary forests, caused increases in soil acidity. Eight-year old A. mangium plantations contained most carbon in biomass+soil, and secondary forests and pastures had similar or higher soil carbon. E. urophylla plantations had the lowest soil carbon status, raising doubt about their sequestration capacity in current 6–8 year rotations.

Conclusions

The study demonstrates that appropriate reforestation enhances soil fertility and promotes carbon sequestration on degraded tropical lands and that unmanaged secondary forests are effective at improving soil fertility and sequestering carbon at low cost.     

The Green Microalga Chlamydomonas reinhardtii Has a Single ω-3 Fatty Acid Desaturase That Localizes to the Chloroplast and Impacts Both Plastidic and Extraplastidic Membrane Lipids

The ω-3 polyunsaturated fatty acids account for more than 50% of total fatty acids in the green microalga Chlamydomonas reinhardtii, where they are present in both plastidic and extraplastidic membranes. In an effort to elucidate the lipid desaturation pathways in this model alga, a mutant with more than 65% reduction in total ω-3 fatty acids was isolated by screening an insertional mutant library using gas chromatography-based analysis of total fatty acids of cell pellets. Molecular genetics analyses revealed the insertion of a TOC1 transposon 113 bp upstream of the ATG start codon of a putative ω-3 desaturase (CrFAD7; locus Cre01.g038600). Nuclear genetic complementation of crfad7 using genomic DNA containing CrFAD7 restored the wild-type fatty acid profile. Under standard growth conditions, the mutant is indistinguishable from the wild type except for the fatty acid difference, but when exposed to short-term heat stress, its photosynthesis activity is more thermotolerant than the wild type. A comparative lipidomic analysis of the crfad7 mutant and the wild type revealed reductions in all ω-3 fatty acid-containing plastidic and extraplastidic glycerolipid molecular species. CrFAD7 was localized to the plastid by immunofluorescence in situ hybridization. Transformation of the crfad7 plastidial genome with a codon-optimized CrFAD7 restored the ω-3 fatty acid content of both plastidic and extraplastidic lipids. These results show that CrFAD7 is the only ω-3 fatty acid desaturase expressed in C. reinhardtii, and we discuss possible mechanisms of how a plastid-located desaturase may impact the ω-3 fatty acid content of extraplastidic lipids.Research on lipid metabolism in microalgae has flourished in recent years due to their potential as a rich source of ω-3 fatty acids (Guschina and Harwood, 2006; Khozin-Goldberg et al., 2011) and as a feedstock for biodiesel (Hu et al., 2008b; Rosenberg et al., 2008; Beer et al., 2009; Radakovits et al., 2010; Wijffels and Barbosa, 2010; Merchant et al., 2012; Work et al., 2012). Oils produced by microalgae resemble that of plants (Hu et al., 2008b), with the exception that they contain higher proportions of polyunsaturated fatty acid (PUFA) species (Harwood and Guschina, 2009). Desaturation of acyl groups in glycerolipids is catalyzed by fatty acid desaturases (FADs), which insert a C=C bond at a specifically defined position of an acyl chain (Shanklin and Cahoon, 1998). The degree of unsaturation of fatty acid components largely determines the chemical property and thus the utility of the oils produced. FADs have been one of the major tools for the genetic engineering of oil composition in land crops (Shanklin and Cahoon, 1998; Napier et al., 1999). In view of biodiesel applications, low PUFA content is advantageous in algal oil because of oxidation issues (Frankel, 1991).With the suites of sophisticated molecular genetic and genomic tools developed in the green microalga Chlamydomonas reinhardtii and the existence of substantial literature related to its cell biology, physiology, and biochemistry, this organism has emerged as a major model for research on algal oil (Radakovits et al., 2010; Merchant et al., 2012; Liu and Benning, 2013). Although the understanding of lipid metabolism in C. reinhardtii largely relies on sequence homologies to other models (Riekhof et al., 2005) and is still rather limited compared with the model plant Arabidopsis (Arabidopsis thaliana; Li-Beisson et al., 2010), functional studies based on mutants have started to provide important insights into the biosynthesis and turnover of membrane and storage lipids in this model alga (Riekhof et al., 2005; Work et al., 2010; Fan et al., 2011; Goodson et al., 2011; Boyle et al., 2012; Li et al., 2012a, 2012b; Yoon et al., 2012).In C. reinhardtii, C16 and C18 PUFAs (ω-3 + ω-6) make up to 60 mol% of total membrane fatty acids, of which more than 80% are ω-3 species (Giroud and Eichenberger, 1988; Siaut et al., 2011). Biochemical evidence for lipid-linked desaturation of fatty acyl chains has been established in C. reinhardtii over 20 years (Giroud and Eichenberger, 1989), but only two C. reinhardtii mutants affected in fatty acid desaturation have been described to date. These are crfad6 (hf-9), an insertional mutant for the plastidial ω-6 desaturase FAD6 (Sato et al., 1995), and microRNA-based silenced lines for the Δ4 desaturase CrΔ4FAD (Zäuner et al., 2012). The putative microsomal Δ12 desaturase FAD2 (Chi et al., 2008) and front-end ω-13 desaturase (Kajikawa et al., 2006) have been characterized by heterologous expression in the methylotrophic yeast Pichia pastoris, but no mutant is available. Moreover, although ω-3 PUFA is the most abundant fatty acid class in C. reinhardtii, the ω-3 desaturase remains uncharacterized, and no mutant with specific reduction in ω-3 content has been isolated so far.In Arabidopsis and C. reinhardtii, ω-3 PUFAs are present in both plastidic and extraplastidic lipids such as monogalactosyldiacylglycerol (MGDG) and phosphatidylethanolamine (PtdEtn), respectively (Mendiola-Morgenthaler et al., 1985; Giroud et al., 1988). While in plants there are distinct genes for plastidial and extraplastidial ω-3 FADs (Wallis and Browse, 2002), only one putative ω-3 desaturase seems encoded in the C. reinhardtii genome (version 5.0; Merchant et al., 2007). This raises several intriguing possibilities, including the existence of a mechanism to export ω-3 acyls from their site of biogenesis to other membranes or a dual localization of the ω-3 desaturase homolog (plastid and endoplasmic reticulum [ER]). In this study, we report the identification and characterization of a C. reinhardtii mutant defective in the promoter region of the putative ω-3 FAD encoded by the Cre01.g038600 locus. We show that while this enzyme is localized to plastids, impairment in its expression leads to a reduction of ω-3 fatty acids acylated to both plastidial and ER lipids. Additionally, using plastidial transformation of the mutant, it is demonstrated that the location of this desaturase in the plastid alone is sufficient to ensure normal ω-3 fatty acid content in extraplastidic lipids. Possible acyl desaturation and trafficking mechanisms implied by these findings are discussed.