Sulfur assimilation by Oxyrrhis marina feeding on a 35S‐DMSP‐labelled prey

A laboratory grazing experiment was conducted with the aim of quantifying the sulfur assimilation by a herbivore protist feeding on a dimethylsulfoniopropionate (DMSP)‐containing phytoplankter. When supplied with dissolved ³⁵S‐DMSP, cultures of an axenic strain of the diatom Thalassiosira pseudonana took up 60–95% of the added radioisotope and accumulated it untransformed in the cytoplasm. Radiolabelled diatom cells were offered as prey to the heterotrophic dinoflagellate Oxyrrhis marina. After 32 h in the dark, all the prey had been grazed and digested, leaving only radiolabelled O. marina in the grazing bottles and thus providing an estimate of the percentage of DMSP‐sulfur retained by the predator. Subsequent precipitation with cold trichloroacetic acid (TCA) provided the fraction of retained DMSP‐S that had been assimilated into the micrograzer macromolecules. In parallel incubations with predator and dissolved ³⁵S‐DMSP only (no prey), O. marina (and their closely associated bacteria) took up the radiolabelled substrate osmotrophically to an activity of 0.04 dpm cell^?1 and assimilated it all into macromolecules. By correcting grazing ³⁵S‐DMSP assimilation for osmotrophic ³⁵S‐DMSP assimilation, and comparing it with the ingested radioisotope, the percentage of ingested DMSP‐sulfur retained and assimilated by the predator was determined to be 32 ± 4%. This is the first study that provides direct evidence that ingestion of a DMSP‐containing prey supplies structural sulfur to a herbivore protist and that quantifies this assimilative supply at one‐third of ingested DMSP.     

Formic Acid and Acetic Acid Induce a Programmed Cell Death in Pathogenic Candida Species

Cutaneous fungal infections are common and widespread. Antifungal agents used for the treatment of these infections often have undesirable side effects. Furthermore, increased resistance of the microorganisms to the antifungal drugs becomes the growing problem. Accordingly, the search for natural antifungal compounds continues to receive attention. Apoptosis is highly regulated programmed cell death. During yeast cell apoptosis, amino acids and peptides are released and can stimulate regeneration of human epithelium cells. Thus, detection of chemical compounds inducing apoptosis in yeast and nontoxic for humans is of great medical relevance. The aim of this study was to detect chemical compound inducing apoptosis in pathogenic Candida species with the lowest toxicity to the mammalian cells. Five chemical compounds—acetic acid, sodium bicarbonate, potassium carbonate, lithium acetate, and formic acid—were tested for evaluation of antifungal activity on C. albicans, C. guilliermondii, and C. lusitaniae. The results showed that acetic acid and formic acid at the lowest concentrations induced yeast cells death. Apoptosis analysis revealed that cells death was accompanied by activation of caspase. Minimal inhibitory concentrations of potassium carbonate and sodium bicarbonate induced Candida cells necrosis. Toxicity test with mammalian cell cultures showed that formic acid has the lowest effect on the growth of Jurkat and NIH 3T3 cells. In conclusion, our results show that a low concentration of formic acid induces apoptosis-like programmed cell death in the Candida yeast and has a minimal effect on the survivability of mammalian cells, suggesting potential applications in the treatment of these infections.     

Chemically Defined Diet Alters the Protective Properties of Fructo-Oligosaccharides and Isomalto-Oligosaccharides in HLA-B27 Transgenic Rats

Non-digestible oligosaccharides (NDO) were shown to reduce inflammation in experimental colitis, but it remains unclear whether microbiota changes mediate their colitis-modulating effects. This study assessed intestinal microbiota and intestinal inflammation after feeding chemically defined AIN-76A or rat chow diets, with or without supplementation with 8 g/kg body weight of fructo-oligosaccharides (FOS) or isomalto-oligosaccharides (IMO). The study used HLA-B27 transgenic rats, a validated model of inflammatory bowel disease (IBD), in a factorial design with 6 treatment groups. Intestinal inflammation and intestinal microbiota were analysed after 12 weeks of treatment. FOS and IMO reduced colitis in animals fed rat chow, but exhibited no anti-inflammatory effect when added to AIN-76A diets. Both NDO induced specific but divergent microbiota changes. Bifidobacteria and Enterobacteriaceae were stimulated by FOS, whereas copy numbers of Clostridium cluster IV were decreased. In addition, higher concentrations of total short-chain fatty acids (SCFA) were observed in cecal contents of rats on rat chow compared to the chemically defined diet. AIN-76A increased the relative proportions of propionate, iso-butyrate, valerate and iso-valerate irrespective of the oligosaccharide treatment. The SCFA composition, particularly the relative concentration of iso-butyrate, valerate and iso-valerate, was associated (P≤0.004 and r≥0.4) with increased colitis and IL-1 β concentration of the cecal mucosa. This study demonstrated that the protective effects of fibres on colitis development depend on the diet. Although diets modified specific cecal microbiota, our study indicates that these changes were not associated with colitis reduction. Intestinal inflammation was positively correlated to protein fermentation and negatively correlated with carbohydrate fermentation in the large intestine.     

Prevalence of Sleep Apnea and Electrocardiographic Disturbances in Morbidly Obese Patients

Objective: To determine the prevalence of sleep apnea in morbidly obese patients and its relationship with cardiac arrhythmias. Research Methods and Procedures: Fifty‐two consecutive morbidly obese (body mass index ≥ 40 kg/m²) outpatients from the Obesity Clinic of the National Institute of Nutrition Salvador Zubirán underwent two nights of polysomnography with standard laboratory techniques. Electrocardiographic polysomnography signals (Lead II) were evaluated by two experienced cardiologists, and sleep complaints were measured with a standard sleep questionnaire (Sleep Disorders Questionnaire). In order to make comparisons between groups with different severities of sleep‐disordered breathing, we classified the patients in four groups using the apnea‐hypopnea index (AHI): Group 1, AHI 5 < 15 (n = 10); Group 2, AHI 15 < 30 (n = 10); Group 3, AHI 30 < 65 (n = 14); Group 4, AHI ≥ 65 (n = 17). Results: A wide range of sleep‐disordered breathing, ranging from AHI of 2.5 to 128.9 was found. Ninety‐eight percent of the sample (n = 51) had an AHI ≥ 5 (mean = 51 ± 37), and 33% had severe sleep apnea with AHI ≥ 65 with a mean nocturnal desaturation time of <65% over 135 minutes. Electrocardiographic abnormalities were present in 31% of the patients. Cardiac rhythm alterations showed an association with the level of sleep‐disordered breathing and oxygen desaturation. Discussion: We conclude that there is a high prevalence of sleep apnea in morbidly obese patients and that the risk for cardiac arrhythmias increases in this population in the presence of a severe sleep apnea (AHI ≥ 65) with severe oxygen desaturation (Sao ₂ ≤ 65%).     

Xylose fermentation as a challenge for commercialization of lignocellulosic fuels and chemicals

Fuel ethanol production from lignocellulosic materials is at a level where commercial biofuel production is becoming a reality. The solubilization of the hemicellulose fraction in lignocellulosic-based feedstocks results in a large variety of sugar mixtures including xylose. However, allowing xylose fermentation in yeast that normally is used for fuel ethanol production requires genetic engineering. Moreover, the efficiency of lignocellulosic pretreatment, together with the release and generation of inhibitory compounds in this step, are some of the new challenges faced during second generation ethanol production. Successful advances in all these aspects will improve ethanol yield, productivity and titer, which will reduce the impact on capital and operating costs, leading to the consolidation of the fermentation of lignocellulosic biomass as an economically feasible option for the production of renewable fuels. Therefore the development of yeast strains capable of fermenting a wide variety of sugars in a highly inhibitory environment, while maintaining a high ethanol yield and production rate, is required. This review provides an overview of the current status in the use of xylose-engineered yeast strains and describes the remaining challenges to achieve an efficient deployment of lignocellulosic-based ethanol production.     

Tryptophan metabolism activation by indoleamine 2,3-dioxygenase in adipose tissue of obese women: an attempt to maintain immune homeostasis and vascular tone

Human obesity is characterized by chronic low-grade inflammation in white adipose tissue and is often associated with hypertension. The potential induction of indoleamine 2,3-dioxygenase-1 (IDO1), the rate-limiting enzyme in tryptophan/kynurenine degradation pathway, by proinflammatory cytokines, could be associated with these disorders but has remained unexplored in obesity. Using immunohistochemistry, we detected IDO1 expression in white adipose tissue of obese patients, and we focused on its contribution in the regulation of vascular tone and on its immunoregulatory effects. Concentrations of tryptophan and kynurenine were measured in sera of 36 obese and 15 lean women. The expression of IDO1 in corresponding omental and subcutaneous adipose tissues and liver was evaluated. Proinflammatory markers and T-cell subsets were analyzed in adipose tissue via the expression of CD14, IL-18, CD68, TNFα, CD3ε, FOXP3 [a regulatory T-cell (Treg) marker] and RORC (a Th17 marker). In obese subjects, the ratio of kynurenine to tryptophan, which reflects IDO1 activation, is higher than in lean subjects. Furthermore, IDO1 expression in both adipose tissues and liver is increased and is inversely correlated with arterial blood pressure. Inflammation is associated with a T-cell infiltration in obese adipose tissue, with predominance of Th17 in the omental compartment and of Treg in the subcutaneous depot. The Th17/Treg balance is decreased in subcutaneous fat and correlates with IDO1 activation. In contrast, in the omental compartment, despite IDO1 activation, the Th17/Treg balance control is impaired. Taken together, our results suggest that IDO1 activation represents a local compensatory mechanism to limit obesity-induced inflammation and hypertension.     

Inflammatory Bowel Disease: How Effective Is TNF-α Suppression?

Crohn’s Disease (CD) results from inappropriate response toward commensal flora. Earlier studies described CD as a Th1 mediated disease. Current models view both phenotypes as a continuum of various permutations between Th1, Th2 and Th17 pathways compounded by a range of Treg disfunctions. In the present paper, we develop a mathematical model, by a system of differential equations, which describe the dynamic relations among these T cells and their cytokines. The model identities four groups of CD patients according to up/down regulation of Th1 and Th2. The model simulations show that immunosuppression by TNF-α blockage benefits the group with Th1^High/Th2^Low while, by contrast, the group with Th1^Low/Th2^High will benefit from immune activation.     

Modulating the Expression Strength of the Baculovirus/Insect Cell Expression System: A Toolbox Applied to the Human Tumor Suppressor SMARCB1/SNF5

The human tumor suppressor SMARCB1/INI1/SNF5/BAF47 (SNF5) is a core subunit of the multi-subunit ATP-dependent chromatin remodeling complex SWI/SNF, also known as Brahma/Brahma-related gene 1 (BRM/BRG1)-associated factor (BAF). Experimental studies of SWI/SNF are currently considerably limited by the low cellular abundance of this complex; thus, recombinant protein production represents a key to obtain the SWI/SNF proteins for molecular and structural studies. While the expression of mammalian proteins in bacteria is often difficult, the baculovirus/insect cell expression system can overcome limitations of prokaryotic expression systems and facilitate the co-expression of multiple proteins. Here, we demonstrate that human full-length SNF5 tagged with a C-terminal 3?×?FLAG can be expressed and purified from insect cell extracts in monomeric and dimeric forms. To this end, we constructed a set of donor and acceptor vectors for the expression of individual proteins and protein complexes in the baculovirus/insect cell expression system under the control of a polyhedrin (polh), p10, or a minimal Drosophila melanogaster Hsp70 promoter. We show that the SNF5 expression level could be modulated by the selection of the promoter used to control expression. The vector set also comprises vectors that encode a 3?×?FLAG tag, Twin-Strep tag, or CBP-3?×?FLAG-TEV-ProteinA triple tag to facilitate affinity selection and detection. By gel filtration and split-ubiquitin assays, we show that human full-length SNF5 has the ability to self-interact. Overall, the toolbox developed herein offers the possibility to flexibly select the promoter strength as well as the affinity tag and is suggested to advance the recombinant expression of chromatin remodeling factors and other challenging proteins.