DESI–MS as a tool for direct lipid analysis in cultured cells

Desorption electrospray ionization may be used as a fast and convenient method for analysis and identification of lipids in the cell culture. Oxidative stress, which usually involves changes in lipids, was used as a model of pathology to show the utility of this analysis methodology. This paper addresses the surface preparation of cell culture slides, induction of oxidative stress, and cell monolayer culture preparation as well as optimization of the analysis. Advantages and drawbacks of the method were also discussed.     

Growth,phenology, and biomass allocation of alien Solidago species in central Europe

Plant invasion is a major threat to the integrity of an ecosystem. Exceptionally successful invaders in Europe are the American species of Solidago genus. In this study we examined growth, reproduction, and phenology of Solidago species, of American origin, growing in central Europe (S. altissima, S. canadensis, S. gigantea, S. graminifolia). These taxa were compared with two native species: Solidago virgaurea and Tanacetum vulgare. We observed high differentiation in height, number of shoots, and biomass production between individuals within taxa. Generally, the invasive species produced substantially (two to five times) more biomass than the native ones, being statistically significant in the comparison of alien Solidago graminifolia and S. gigantea versus native S. virgaurea and T. vulgare. The ratio of biomass of reproductive parts to overall biomass varied considerably among years, but generally the lowest one was for Solidago altissima, and the highest for S. graminifolia. It shows a lack of a clear pattern of differentiation between alien and native species in terms of biomass investment in reproduction. We observed a general tendency of allocation of a major part of biomass in rhizomes by phalanx species (S. graminifolia and S. gigantea), while species with guerrilla strategy (S. altissima and S. canadensis) invested more biomass in stems and leaves. However, because of the high variability there was no clear, stable pattern of statistically significant differences between these two groups. The results suggest that S. graminifolia reveals a strong potential of invasion, in spite of its, so far limited, distribution in Europe.     

Quantification of Alternaria,Cladosporium, Fusarium and Penicillium verrucosum in Conventional and Organic Grains by qPCR

We analysed the levels of Alternaria, Cladosporium, Fusarium and Penicillium verrucosum in grain samples harvested in 2011 and 2012 from conventional and organic farms using qPCR. In general, both Alternaria and Cladosporium occurred in all cereal grains in the highest quantities, followed by P. verrucosum and Fusarium. Alternaria, Cladosporium and P. verrucosum had the highest levels in crop mixtures, barley and rye and lower levels in wheat, while Fusarium levels were the highest in crop mixtures and wheat. The levels of Alternaria and P. verrucosum were higher in organic rye and wheat than conventional grains. Although the level of Fusarium was higher in conventional than organic rye, opposite results were obtained for crop mixtures. A positive correlation was found between Alternaria, Cladosporium and P. verrucosum, indicating that similar factors might affect the distribution of these fungi in grains.     

Dendritic cells are stressed out in tumor

A recently paper published in Cell reports that dendritic cells (DCs) are dysfunctional in the tumor environment. Tumor impairs DC function through induction of endoplasmic reticulum stress response and subsequent disruption of lipid metabolic homeostasis.Tumors develop diverse strategies to escape tumor-specific immunity. Tumor-infiltrating dendritic cells (tDCs) are dysfunctional and/or mediate immune suppression¹. Cubillos-Ruiz et al.² showed in their recent Cell paper that tDCs exhibit an activation of the unfolded protein response (UPR), as indicated by the presence of high levels of spliced XPB1, and this may be attributed to reactive oxygen species (ROS) in tumor, which induces lipid peroxidation, leading to the endoplasmic reticulum (ER) stress in tDCs. Furthermore, they demonstrated that UPR activation in tDCs results in poor DC function, which is accompanied by impaired lipid metabolism and subsequent reduction of T cell anti-tumor immunity. Thus, these observations present a novel mechanism for tDC malfunction.ER stress is evoked by the presence of unfolded or chemically modified proteins. In short, the presence of damaged proteins is sensed by the proteins in the ER membrane. Of these, IRE1α can remove a short nucleotide sequence from mRNA encoding XBP1 protein. This splicing event facilitates XBP1 translation. XBP1 protein binds to consensus sequences in target genes and activates their expression³. Many XBP1 target genes are fatty acid synthesis enzymes. Enhanced production of fatty acids leads to the formation of lipid droplets inside the cytoplasm and extension of the ER compartment due to efficient intracellular membrane formation³. Therefore, this mechanism is a form of adaptation of the cell to the harsh environment, which sustains the production of functional proteins. In such a context, the article by Cuillos-Ruiz et al.² shows intriguing data on the XBP1 pathway in silencing DC function in the tumor environment.First, Cubillos-Ruiz et al.² observed that tDCs express high levels of spliced XBP1, its direct target genes and other markers for the ER stress response. Targeted deletion of XBP1 in CD11c⁺ DCs reveals an association of the XBP1-dependent ER stress response with immunosuppressive properties of tDCs. DC-specific deletion of XBP1 not only inhibits tumor growth and prolongs animal survival, but also reduces tumor peritoneal metastasis, ascites accumulation, and splenomegaly in an ovarian cancer-bearing mouse model.Next, the authors elucidated why the XBP1 pathway in tDCs is highly activated. Unexpectedly, neither typical tumor-associated cytokines nor hypoxia can efficiently stimulate XBP1 activation. Interestingly, tDCs contain high levels of lipid peroxidation byproducts bound to the proteins, which is associated with the production of ROS. Microarray analysis revealed that DC-specific XBP1 deletion downregulates both UPR pathway-dependent genes and lipid metabolism, which results in lower total lipid production, loss of lipid droplets in the cytoplasm and decreased production of triacylglycerides. This phenotype can be recapitulated by chemical inhibition of ROS formation or IRE1α and XBP1 signaling. Furthermore, XBP1-deficient DCs are potent stimulators of OT-1 T cells. Adoptive transfer of T cells isolated from metastatic tumor-bearing mice with DC-specific XBP1 deletion also shows their superior ability to control tumor growth.On the basis of these observations, the authors tested the effects of therapeutic intervention with the usage of nanocomplexes containing XBP1 siRNA. The size of lipid particles or nanocomplexes determines the anatomical location of specific drug delivery⁴. Additionally, they previously optimized the nanocomplexes for selective engulfing by DCs⁵. They found that administration of the nanoparticles containing XBP1 siRNA causes potent T cell activation, which is accompanied by reduced cancer metastatic foci and improved animal survival.The paper by Cubillos-Ruiz et al.² provides an important insight into DC biology in general. Since the identification of Toll-like receptor (TLR) signaling, the main direction in DC biology has been associated with PAMP and DAMP recognition in various degrees (Figure 1). However, the link between cellular metabolism in specific microenvironment and DC biology is poorly explored (Figure 1). The ER stress response has previously been observed in DCs^6,7. It is thought that in response to ER stress, XBP1 is crucial for DC generation, survival, and function^6,7. The conceptual link between XBP1 signaling and DC biology is as follows⁸: (i) stimulation of DCs leads to ER stress; (ii) ER stress activates UPR/XBP1 pathway; (iii) XBP1 activates lipid synthesis genes; (iv) lipids are used for the extension of the ER and Golgi compartment, which are of importance for cytokine production and secretion^9,10. Now, the authors challenged this concept in the context of cancer. The authors demonstrated that XBP1 signaling is strongly associated with poor T cell activation and limitation of XBP1 signaling leads to improved T cell function in the tumor environment (Figure 1). Thus, the paper sheds a new light on DC biology.Open in a separate windowFigure 1Double-faced role of XBP1 signaling pathway in DCs. Left: under immunostimulatory conditions, DCs receive signals from Toll-like receptors. NF-κB signaling induces a XBP1-dependent ER stress response, which enhances lipid metabolism. Formation of new membranes expands ER and Golgi compartments, which enhances cytokine production and secretion⁷. Right: In the tumor microenvironment, DCs are exposed to ROS, which also results in ER stress. However, in this case DC lipid metabolism is impaired and DCs acquire an immunosuppressive phenotype².Of course, as with any interesting work, the article raises more questions than answers. For example, which immune-related properties of DCs are “selectively” affected by the XBP1 pathway? Is there an actual cause-and-effect relationship between aberrant lipid accumulation and the immunosuppressive phenotype of tDCs? The authors observed no obvious change in PD-L1 (B7-H1) expression in DCs, but noticed reduced surface levels of peptide-loaded MHC-I complexes in wild-type tDCs as compared to XBP-deficient tDCs. However, it remains unknown whether and how DC cross-presentation is involved in tDC function regulated by ER stress, XBP1 activation, and lipid metabolism. Human ovarian cancer-associated DCs express high levels of B7-H1 and limited IL-12¹¹. It would be interesting to thoroughly examine the cytokine profile, and the B7 and TNF family members, along with lipid pathway manipulation in XBP1^−/− DCs. It is well known that ER morphology and function is substantial for the synthesis of membrane proteins and cytokines, which would be affected by the XBP1 pathway. Paradoxically, a restricted ER stress response can help immune reaction, which requires the expansion of the ER compartment. Another question is how and which lipid synthesis and metabolism pathway is targeted by XBP1 in DCs (or/and tumor cells in the same environment). Obviously, future studies are warranted to address these important questions.In conclusion, the paper by Cubillos-Ruiz et al.² opens an interesting chapter for scientifically and therapeutically exploring DC biology in a specific metabolic environment. Given that silencing XBP1 signaling in DCs enhances tumor immunity, and XBP1 is an intrinsic pro-tumor factor, it is reasonable to assume that targeting this pathway may be beneficial in patients with cancer and could kill two birds with one stone.     

Design and synthesis of new substrates of HtrA2 protease

HtrA2 belongs to the HtrA (high temperature requirement A) family of ATP-independent serine proteases. The primary function of HtrA2 includes maintaining the mitochondria homeostasis, cell death (by apoptosis, necrosis, or anoikis), and contribution to the cell signaling. Several recent reports have shown involvement of HtrA2 in development of cancer and neurodegenerative disorders. Here, we describe the profiling of HtrA2 protease substrate specificity via the combinatorial chemistry approach that led to the selection of novel intramolecularly quenched substrates. For all synthesized compounds, the highest HtrA2-mediated hydrolysis efficiency and selectivity among tested HtrA family members was observed for ABZ-Ile-Met-Thr-Abu-Tyr-Met-Phe-Tyr(3-NO₂)-NH₂, which displayed a specificity constant k_cat/K_M value of 14,535 M⁻¹ s⁻¹.     

Memantine reduces consumption of highly palatable food in a rat model of binge eating

Excessive consumption of highly palatable food has been linked to the development of eating disorders and obesity, and can be modeled in non-food-deprived rats by offering them a limited (2-h daily) access to an optional dietary fat. Since the glutamatergic system has recently emerged as a viable target for binge-eating medication development, we compared the effects of subchronic treatment with glutamatergic receptor antagonists to the effects of a reference appetite-suppressing agent sibutramine on highly palatable food (lard) and normal chow intake. In three separate experiments, the consumption of a standard laboratory chow and lard were measured during 12 days of medication treatment and for 6 days afterwards. Generalized estimating equations analysis demonstrated that sibutramine (7.5 mg/kg, PO) significantly decreased lard consumption, with a concurrent increase in chow consumption. Sibutramine effects disappeared after treatment discontinuation. The NMDA receptor antagonist memantine (5 mg/kg, IP) significantly decreased lard consumption and increased chow consumption, comparable to effects of sibutramine; however, memantine’s effects persisted after treatment discontinuation. The effects of the mGluR5 antagonist MTEP (7.5 mg/kg, IP) on food consumption were in the same direction as seen with memantine, but the observed differences were not significant. In an additional control experiment, sibutramine and memantine reduced unlimited (24 h) chow intake during the treatment phase. Present results provide evidence that glutamatergic neurotransmission might be involved in the regulation of excessive consumption of highly palatable foods, and suggest that NMDA receptor may be an attractive target for developing obesity and disordered eating pharmacotherapies.     

Ultrafiltrative separation of rhamnolipid from culture medium

Classic methods of biosurfactant separation are difficult and require large amounts of organic solvents, thus generate high amounts of waste. This work presents and discusses in detail an original procedure to separate rhamnolipid from fermentation broth using high performance membrane techniques. Due to the unique properties of surface active agents, such as capability of forming aggregates above the critical micelle concentration, it is possible to easily purify the biosurfactant with high efficacy using inexpensive and commonly used membranes. In this article, two-stage ultrafiltration is proposed as a method for separating and purifying rhamnolipid from the culture medium. The obtained purified rhamnolipid solution was capable of reducing surface tension of water down to 28.6 mN/m at critical micelle concentration of 40 mg/l. Separation of rhamnolipid was confirmed by HPLC; three types of rhamnolipids were identified (RL1, RL2, RL4), with considerable predominance of RL2.     

Synthesis and in vitro study of antiviral and virucidal activity of novel 2-[(4-methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetamide derivatives

2-[(4-Methyl-4H-1,2,4-triazol-3-yl)sulfanyl]acetamide derivatives were synthesized and their structures were confirmed by 1H NMR, IR, and elemental analysis. Cytotoxicity of the compounds towards HEK-293 and GMK cells was evaluated. Moreover, the antiviral and virucidal activities of these compounds against human adenovirus type 5 and ECHO-9 virus were assessed. Some of the newly synthesized derivatives have the potential to reduce the viral replication of both tested viruses.