The Influence of Two Lithium Forms on the Growth, l-Ascorbic Acid Content and Lithium Accumulation in Lettuce Plants

Lithium (Li) is a trace element that is essential in the human diet due to its importance for health and proper functioning of an organism. However, the biological activity of this metal in crop plants, which are the primary dietary sources of Li, is still poorly understood. The aim of the presented study was to comparatively analyse two Li chemical forms on the growth, as well as the l-ascorbic acid content, the Li accumulation and translocation in butterhead lettuce (Lactuca sativa L. var. capitata) cv. Justyna. The plants were grown in a nutrient solution enriched with Li in the form of LiCl or LiOH at the following concentrations: 0, 2.5, 20, 50 or 100 mg?Li?dm^?3. The obtained results indicate that the presence of Li⁺ ions in the root environment reduced the yield of edible parts of the lettuce if the Li concentration in a nutrient solution had reached 20 mg?Li?dm^?3. However, a yield reduction under these conditions was found to be significant only for LiOH. In plants exposed to 50 mg?Li?dm^?3, both shoot and root fresh weights (FW) significantly decreased, regardless of the supplied Li chemical form. On the other hand, under the lowest LiOH dose, a significant increase in the root FW was noted, suggesting beneficial effects of Li on the growth of lettuce plants. However, applied Li concentrations and forms did not affect the l-ascorbic acid content in the lettuce leaves. Regardless of which Li form was used, Li accumulated mainly in the root tissues. An exception was the higher concentration of this metal in the shoots than in the roots of plants supplied with 100 mg?Li?dm^?3 in LiCl, and there were almost the same Li concentrations in both examined organs of plants supplied with 100 mg?Li?dm^?3 in LiOH. The effectiveness of Li translocation from roots to shoots rose with increasing Li concentrations in the growth medium, and this suggests a relatively ready translocation of this metal throughout the plant. Moreover, these results suggest that Li toxicity in lettuce plants is related to a high accumulation of this element in the root and shoot tissues, causing a drastic reduction in the yield, in the presence either of LiCl or LiOH, but not affecting the l-ascorbic acid accumulation in the leaves.     

Iron Excess Disturbs Metabolic Status and Relative Gonad Mass in Rats on High Fat, Fructose, and Salt Diets

The aim of this study was to assess the metabolic and physiological changes in rats fed a diet high in fat, fructose, and salt, and with excess iron level. Mineral status was also estimated. Wistar rats were assigned to groups fed either a standard control diet (C) or a diet high in fat, fructose, and salt. The noncontrol diets contained either normal (M) or high level (MFe) of iron. After 6 weeks, the length and weight of the rats were measured, and the animals were euthanized. The kidneys and gonads were collected, and blood samples were taken. Serum levels of insulin, nitric oxide, and iron were measured. The iron, zinc, copper, and calcium concentrations of tissues were determined. It was found that the M diet led to a significant increase in the relative kidney mass of the rats compared with the control group. Among the rats fed the M diet, markedly higher serum level of iron and lower levels of zinc and copper were observed in tissues, while significantly higher calcium levels were found in the gonads. The MFe diet resulted in decreased obesity index, insulin level, and nitric oxide serum concentration in the rats, when compared with both the M and C diets. The high iron level in the modified diet increased the relative mass of the gonads. The excess iron level in the diet disturbed the zinc, copper, and calcium status of tissues. The decrease in insulin and nitric oxide in rats fed the diet high in iron, fat, fructose, and salt was associated with disorders of zinc, copper, and calcium status, as well as with an increase in the relative mass of the gonads.     

FOXO1 promotes wound healing through the up-regulation of TGF-β1 and prevention of oxidative stress

Keratinocyte mobilization is a critical aspect of wound re-epithelialization, but the mechanisms that control its precise regulation remain poorly understood. We set out to test the hypothesis that forkhead box O1 (FOXO1) has a negative effect on healing because of its capacity to inhibit proliferation and promote apoptosis. Contrary to expectations, FOXO1 is required for keratinocyte transition to a wound-healing phenotype that involves increased migration and up-regulation of transforming growth factor β1 (TGF-β1) and its downstream targets, integrin-α3 and -β6 and MMP-3 and -9. Furthermore, we show that FOXO1 functions in keratinocytes to reduce oxidative stress, which is necessary to maintain cell migration and prevent cell death in a TGF-β1–independent manner. Thus, our studies identify a novel function for FOXO1 in coordinating the response of keratinocytes to wounding through up-regulation of TGF-β1 and other factors needed for keratinocyte migration and protection against oxidative stress, which together promote migration and decrease apoptosis.     

Mechanism of Bacterial Oligosaccharyltransferase: IN VITRO QUANTIFICATION OF SEQUON BINDING AND CATALYSIS*

N-Linked glycosylation is an essential post-translational protein modification in the eukaryotic cell. The initial transfer of an oligosaccharide from a lipid carrier onto asparagine residues within a consensus sequon is catalyzed by oligosaccharyltransferase (OST). The first X-ray structure of a complete bacterial OST enzyme, Campylobacter lari PglB, was recently determined. To understand the mechanism of PglB, we have quantified sequon binding and glycosylation turnover in vitro using purified enzyme and fluorescently labeled, synthetic peptide substrates. Using fluorescence anisotropy, we determined a dissociation constant of 1.0 μm and a strict requirement for divalent metal ions for consensus (DQNAT) sequon binding. Using in-gel fluorescence detection, we quantified exceedingly low glycosylation rates that remained undetected using in vivo assays. We found that an alanine in the −2 sequon position, converting the bacterial sequon to a eukaryotic one, resulted in strongly lowered sequon binding, with in vitro turnover reduced 50,000-fold. A threonine is preferred over serine in the +2 sequon position, reflected by a 4-fold higher affinity and a 1.2-fold higher glycosylation rate. The interaction of the +2 sequon position with PglB is modulated by isoleucine 572. Our study demonstrates an intricate interplay of peptide and metal binding as the first step of protein N-glycosylation.     

Endogenous hormone profiles during early seed development of C. arietinum and C. anatolicum

Cicer anatolicum, a perennial species, has ascochyta blight resistance superior to that found in the cultivated chickpea. However, hybridization barriers during early stages of embryo development curtail access to this trait. Since hormones play an essential role in early embryo development, we have determined the hormone profiles of 4-, 8-, and 12-day old seeds from a Canadian chickpea (Cicer arietinum L.) cv. CDC Xena, from Indian cvs. Swetha and Bharati, and from a perennial accession of C. anatolicum (PI 383626). Indole-3-acetic acid content peaked on day 4 in CDC Xena, on day 8 in both Indian cultivars but only on day 12 in C. anatolicum. The cytokinins, isopentenyladenosine (iPA) and trans zeatin riboside (tZR) were predominant in CDC Xena and Swetha seeds on day 4, whereas cis zeatin riboside was the major component in Bharati. In C. anatolicum, iPA maxed out on day 4 and tZR on day 12. The bioactive gibberellin GA₁ spiked on day 4 in CDC Xena and Bharati, on day 8 in Swetha but only on day 12 in C. anatolicum. Eight-day old seeds had the highest abscisic acid content in the cultivars but spiked on day 12 in the perennial species. The hormone profiles of the perennial species showed delayed spikes in all four hormone groups indicating that there is a mismatch in the hormone requirements of the different embryos. Improving synchronization of early seed hormone profiles of cultivated and perennial chickpea should improve interspecific hybrid production.     

High cell density cultivation of a recombinant E. coli strain expressing a key enzyme in bioengineered heparin production

A bioengineered heparin, as a replacement for animal-derived heparin, is under development that relies on the fermentative production of heparosan by Escherichia coli K5 and its subsequent chemoenzymatic modification using biosynthetic enzymes. A critical enzyme in this pathway is the mammalian 6-O-sulfotransferase (6-OST-1) which specifically sulfonates the glucosamine residue in a heparin precursor. This mammalian enzyme, previously cloned and expressed in E. coli, is required in kilogram amounts if an industrial process for bioengineered heparin is to be established. In this study, high cell density cultivation techniques were exploited to obtain recombinant 6-OST-1. Physiological studies were performed in shake flasks to establish optimized growth and production conditions. Induction strategies were tested in fed-batch experiments to improve yield and productivity. High cell density cultivation in 7-l culture, together with a coupled inducer strategy using isopropyl β-d-1-thiogalactopyranoside and galactose, afforded 482 mg?l^?1 of enzyme with a biomass yield of 16.2 mg?g_cdw ^?1 and a productivity of 10.5 mg?l^?1?h^?1.     

Interspecies distances between propionic acid degraders and methanogens in syntrophic consortia for optimal hydrogen transfer

A mixed culture from an anaerobic biowaste digester was enriched on propionate and used to investigate interspecies hydrogen transfer in dependence of spatial distances between propionate degraders and methanogens. From 20.3 mM propionate, 20.8 mM acetate and 15.5 mM methane were formed. Maximum specific propionate oxidation and methane formation rates were 49 and 23 mmol?mg^?1?day^?1, respectively. Propionate oxidation was inhibited by only 20 mM acetate by about 50 %. Intermediate formate formation during inhibited methanogensis was observed. The spatial distribution and the biovolume fraction of propionate degraders and of methanogens in relation to the total population during aggregate formation were determined. Measurements of interbacterial distances were conducted with fluorescence in situ hybridization by application of group-specific 16S rRNA-targeted probes and 3D image analyses. With increasing incubation time, floc formation and growth up to 54 μm were observed. Propionate degraders and methanogens were distributed randomly in the flocs. The methanogenic biovolume fraction was high at the beginning and remained constant over 42 days, whereas the fraction of propionate degraders increased with time during propionate feeding. Interbacterial distances between propionate degraders and methanogens decreased with time from 5.30 to 0.29 μm, causing an increase of the maximum possible hydrogen flux from 1.1 to 10.3 nmol?ml^?1?min^?1. The maximum possible hydrogen flux was always higher than the hydrogen formation and consumption rate, indicating that reducing the interspecies distance by aggregation is advantageous in complex ecosystems.     

Cathepsin L silencing enhances arsenic trioxide mediated in vitro cytotoxicity and apoptosis in glioblastoma U87MG spheroids

Despite improved treatment options, glioblastoma multiforme (GBM) remains the most aggressive brain tumour with the shortest post-diagnostic survival. Arsenite (As₂O₃) is already being used in the treatment of acute promyelocytic leukaemia (APL), yet its effects on GBM have not been evaluated in detail. In U87MG cell monolayers, we have previously shown that arsenite cytotoxicity significantly increases upon transient inhibition of lysosomal protease Cathepsin L (CatL). As multicellular spheroids more closely represent in vivo tumours, we aimed to evaluate the impact of permanent CatL silencing on arsenite treatment in U87MG spheroids. CatL was stably silenced using shRNA expression plasmid packed lentiviruses. By using metabolic- and cell viability assays, we demonstrated that long-term CatL silencing significantly increased arsenite cytotoxicity in U87MG spheroids. Silenced CatL also increased arsenite-mediated apoptosis in spheroids via elevated p53 expression, Bax/Bcl2 ratio and caspase 3/7 activity, though with lower efficacy than in monolayers. Arsenite cytotoxicity was enhanced by lower CatL activity, since similar cytotoxicity increase was also observed using the novel CatL inhibitor AT094. The results have significant translational impact, since stable CatL silencing would enable the application of lower systemic doses of arsenite to achieve the desired cytotoxic effects on GBMs in vivo.     

DNA barcoding survey of Trichoderma diversity in soil and litter of the Colombian lowland Amazonian rainforest reveals Trichoderma strigosellum sp. nov. and other species

The diversity of Trichoderma (Hypocreales, Ascomycota) colonizing leaf litter as well as the rhizosphere of Garcinia macrophylla (Clusiaceae) was investigated in primary and secondary rain forests in Colombian Amazonia. DNA barcoding of 107 strains based on the internal transcribed spacers 1 and 2 (ITS1 and 2) of the ribosomal RNA gene cluster and the partial sequence of the translation elongation factor 1 alpha (tef1) gene revealed that the diversity of Trichoderma was dominated (71 %) by three common cosmopolitan species, namely Trichoderma harzianum sensu lato (41 %), Trichoderma spirale (17 %) and Trichoderma koningiopsis (13 %). Four ITS 1 and 2 phylotypes (13 strains) could not be identified with certainty. Multigene phylogenetic analysis and phenotype profiling of four strains with an ITS1 and 2 phylotype similar to Trichoderma strigosum revealed a new sister species of the latter that is described here as Trichoderma strigosellum sp. nov. Sequence similarity searches revealed that this species also occurs in soils of Malaysia and Cameroon, suggesting a pantropical distribution.