Engineering two species of yeast as cell factories for 2'-fucosyllactose.

Oligosaccharides present in human breast milk have been linked to beneficial effects on infant health. Inclusion of these human milk oligosaccharides (HMOs) in infant formula can recapitulate these health benefits. As a result, there is substantial commercial interest in a cost-effective source of HMOs as infant formula ingredients. Here we demonstrate that the yeast species Saccharomyces cerevisiae and Yarrowia lipolytica both can be engineered to produce 2'-fucosyllactose (2'FL), which is the most abundant oligosaccharide in human breast milk, at high titer and productivity. Both yeast species were modified to enable uptake of lactose and synthesis of GDP-fucose - the two precursors of 2'FL - by installing a lactose transporter and enzymes that convert GDP-mannose to GDP-fucose. Production of 2'FL was then enabled by expression of α-1,2-fucosyltransferases from various organisms. By screening candidate transporters from a variety of sources, we identified transporters capable of exporting 2'FL from yeast, which is a key consideration for any biocatalyst for 2'FL production. In particular, we identified CDT2 from Neurospora crassa as a promising target for further engineering to improve 2'FL efflux. Finally, we demonstrated production of 2'FL in fermenters at rates and titers that indicate the potential of engineered S. cerevisiae and Y. lipolytica strains for commercial 2'FL production.

Dietary acid load and bone turnover during long-duration spaceflight and bed rest.

Background: Bed rest studies document that a lower dietary acid load is associated with lower bone resorption. Objective: We tested the effect of dietary acid load on bone metabolism during spaceflight. Design: Controlled 4-d diets with a high or low animal protein-to-potassium (APro:K) ratio (High and Low diets, respectively) were given to 17 astronauts before and during spaceflight. Each astronaut had 1 High and 1 Low diet session before flight and 2 High and 2 Low sessions during flight, in addition to a 4-d session around flight day 30 (FD30), when crew members were to consume their typical in-flight intake. At the end of each session, blood and urine samples were collected. Calcium, total protein, energy, and sodium were maintained in each crew member's preflight and in-flight controlled diets. Results: Relative to preflight values, N-telopeptide (NTX) and urinary calcium were higher during flight, and bone-specific alkaline phosphatase (BSAP) was higher toward the end of flight. The High and Low diets did not affect NTX, BSAP, or urinary calcium. Dietary sulfur and age were significantly associated with changes in NTX. Dietary sodium and flight day were significantly associated with urinary calcium during flight. The net endogenous acid production (NEAP) estimated from the typical dietary intake at FD30 was associated with loss of bone mineral content in the lumbar spine after the mission. The results were compared with data from a 70-d bed rest study, in which control (but not exercising) subjects' APro:K was associated with higher NTX during bed rest. Conclusions: Long-term lowering of NEAP by increasing vegetable and fruit intake may protect against changes in loss of bone mineral content during spaceflight when adequate calcium is consumed, particularly if resistive exercise is not being performed. This trial was registered at clinicaltrials.gov as NCT01713634.

Leaf mechanical resistance in plant trait databases: comparing the results of two common measurement methods.

BACKGROUND AND AIMS: The influence of leaf mechanical properties on local ecosystem processes, such as trophic transfer, decomposition and nutrient cycling, has resulted in a growing interest in including leaf mechanical resistance in large-scale databases of plant functional traits. 'Specific work to shear' and 'force to tear' are two properties commonly used to describe mechanical resistance (toughness or strength) of leaves. Two methodologies have been widely used to measure them across large datasets. This study aimed to assess correlations and standardization between the two methods, as measured by two widely used apparatuses, in order to inter-convert existing data in those global datasets. METHODS: Specific work to shear (W(SS)) and force to tear (FT) were measured in leaves of 72 species from south-eastern Australia. The measurements were made including and excluding midribs. Relationships between the variables were tested by Spearman correlations and ordinary least square regressions. KEY RESULTS: A positive and significant correlation was found between the methods, but coefficients varied according to the inclusion or exclusion of the midrib in the measurements. Equations for prediction varied according to leaf venation pattern. A positive and significant (r = 0·90, P < 0·0001) correlation was also found between W(SS) values for fresh and rehydrated leaves, which is considered to be of practical relevance. CONCLUSIONS: In the context of broad-scale ecological hypotheses and used within the constraints recommended here, leaf mechanical resistance data obtained with both methodologies could be pooled together into a single coarser variable, using the equations provided in this paper. However, more detailed datasets of FT cannot be safely filled in with estimations based on W(SS), or vice versa. In addition, W(SS) values of green leaves can be predicted with good accuracy from W(SS) of rehydrated leaves of the same species.

Evolutionary divergence of leaf width and its correlates.

UNLABELLED: • PREMISE OF THE STUDY: The question why leaf dimensions vary so much between species has long puzzled ecologists. Presumably, variation arises from selective forces acting on leaf function but which selective forces and which leaf functions? This investigation assesses the consistency of divergence in plant traits and habitat variables in association with leaf width divergence in the flora of NSW, Australia.• METHODS: More than 80 traits and habitat variables were measured for 25 independent evolutionary divergence events (PICs). Each PIC was represented by two related plant species that had diverged substantially in leaf width. Outgroup species provided indications of the direction of divergence. Most PICs were within genus, so divergences represent relatively recent evolutionary events.• KEY RESULTS: No plant traits or habitat variables were 100% consistently associated with a divergence in leaf width, and surprisingly few diverged in a consistent direction significantly more than what might be expected by chance. This surprising lack of consistent divergence with leaf width contrasted with the result that many of these traits and habitat variables were correlated with leaf width across all species in our data set and in line with correlations reported from other studies. Subcategorizing PICs according to the probable direction of leaf width divergence did not improve consistency.• CONCLUSIONS: These results indicate that evolutionarily recent leaf width divergence events are not tightly tied to divergences in other leaf traits or in environmental situations, despite the broad correlations that have been observed across many species. Rather, cross species correlations are underpinned by earlier divergence events in the phylogeny.

Hsp90 inhibition enhances PI-3 kinase inhibition and radiosensitivity in glioblastoma.

PURPOSE: Combined targeting with a PI3-kinase inhibitor, BKM120, and an Hsp90 inhibitor, HSP990, was investigated as a multi-targeted approach to potentiate cell death in glioblastoma (GBM). Additionally, the effect of dual drug treatment combined with cytotoxic stress (radiation therapy) was examined. METHODS: Four human GBM cell lines containing wild-type or mutated PTEN and/or p53 were studied. The effects of drug treatments on cell viability, apoptosis induction, pAKt activity, cell cycle arrest, clonogenicity, and tumor growth delay were studied. RESULTS: Combined concurrent treatment with both drugs produced more cell killing in cell viability and apoptosis assays than either drug alone. BKM120 plus HSP990 induced suppression of baseline Akt signaling as well as radiation (RT)-induced pAkt signaling in all cell lines. Cell cycle analysis revealed that HSP990 and BKM120, singly or combined, induced G2/M arrest leading to apoptosis/necrosis and polyploidy. Additionally, the drugs radiosensitized GBM cells in clonogenic assays. In vivo tumor growth delay studies demonstrated the effectiveness of combined drug treatment with HSP990 and BKM120 over single drug treatment, as well as the effectiveness of combined drug treatment in enhancing the effectiveness of radiation therapy. CONCLUSIONS: In conclusion, HSP990 and BKM120, with and without RT, are active agents against glioma tumors. The sensitivity to these agents does not appear to depend on PTEN/p53status in the cell lines tested. We suggest that the combined action of both drugs is a viable multi-targeted strategy with the potential to improve clinical outcome for patients with high-grade glioma.

Epidermal growth factor receptor mutation status and rad51 determine the response of glioblastoma to multimodality therapy with cetuximab, temozolomide, and radiation.

PURPOSE: EGFR amplification and mutation (i.e., EGFRvIII) are found in 40% of primary GBM tumors and are believed to contribute to tumor development and therapeutic resistance. This study was designed to investigate how EGFR mutational status modulates response to multimodality treatment with cetuximab, an anti-EGFR inhibitor, the chemotherapeutic agent, temozolomide (TMZ), and radiation therapy (RT). METHODS AND MATERIALS: In vitro and in vivo experiments were performed on two isogenic U87 GBM cell lines: one overexpressing wildtype EGFR (U87wtEGFR) and the other overexpressing EGFRvIII (U87EGFRvIII). RESULTS: Xenografts harboring EGFRvIII were more sensitive to TMZ alone and TMZ in combination with RT and/or cetuximab than xenografts expressing wtEGFR. In vitro experiments demonstrated that U87EGFRvIII-expressing tumors appear to harbor defective DNA homologous recombination repair in the form of Rad51 processing. CONCLUSION: The difference in sensitivity between EGFR-expressing and EGFRvIII-expressing tumors to combined modality treatment may help in the future tailoring of GBM therapy to subsets of patients expressing more or less of the EGFR mutant.

Dietary support of long-duration head-down bed rest.

INTRODUCTION: Dietary control and nutrient intake are critical aspects of any metabolic study, but this is especially true in the case of bed rest studies. We sought to define nutritional requirements, develop menus, and implement them during long-duration head-down bed rest studies. METHODS: The dietary goals were to provide 100% of subjects' nutrient requirements and to maintain subjects' bodyweight to within 3% of their weight on the third day of head-down bed rest. The research dietitian and metabolic kitchen staff are an important part of the multidisciplinary team required to implement a bed rest study. RESULTS: We report herein the planning steps and nutrient intake results from 13 subjects. We also provide insight into some of the dietary challenges that arise during long-duration bed rest studies. DISCUSSION: Regardless of the overall objective of the bed rest study to be performed, nutrition must be carefully planned, implemented, and monitored to prevent results from being compromised.

The nutritional status of astronauts is altered after long-term space flight aboard the International Space Station.

Defining optimal nutrient requirements is critical for ensuring crew health during long-duration space exploration missions. Data pertaining to such nutrient requirements are extremely limited. The primary goal of this study was to better understand nutritional changes that occur during long-duration space flight. We examined body composition, bone metabolism, hematology, general blood chemistry, and blood levels of selected vitamins and minerals in 11 astronauts before and after long-duration (128-195 d) space flight aboard the International Space Station. Dietary intake and limited biochemical measures were assessed during flight. Crew members consumed a mean of 80% of their recommended energy intake, and on landing day their body weight was less (P = 0.051) than before flight. Hematocrit, serum iron, ferritin saturation, and transferrin were decreased and serum ferritin was increased after flight (P < 0.05). The finding that other acute-phase proteins were unchanged after flight suggests that the changes in iron metabolism are not likely to be solely a result of an inflammatory response. Urinary 8-hydroxy-2'-deoxyguanosine concentration was greater and RBC superoxide dismutase was less after flight (P < 0.05), indicating increased oxidative damage. Despite vitamin D supplement use during flight, serum 25-hydroxycholecalciferol was decreased after flight (P < 0.01). Bone resorption was increased after flight, as indicated by several markers. Bone formation, assessed by several markers, did not consistently rise 1 d after landing. These data provide evidence that bone loss, compromised vitamin D status, and oxidative damage are among critical nutritional concerns for long-duration space travelers.

Nutritional status changes in humans during a 14-day saturation dive: the NASA Extreme Environment Mission Operations V project.

Ground-based analogs of spaceflight are an important means of studying physiologic and nutritional changes associated with space travel, and the NASA Extreme Environment Mission Operations V (NEEMO) is such an analog. To determine whether saturation diving has nutrition-related effects similar to those of spaceflight, we conducted a clinical nutritional assessment of the NEEMO crew (4 men, 2 women) before, during, and after their 14-d saturation dive. Blood and urine samples were collected before, during, and after the dive. The foods consumed by the crew were typical of the spaceflight food system. A number of physiologic changes were observed, during and after the dive, that are also commonly observed during spaceflight. Hemoglobin and hematocrit were lower (P < 0.05) after the dive. Transferrin receptors were significantly lower immediately after the dive. Serum ferritin increased significantly during the dive. There was also evidence indicating that oxidative damage and stress increased during the dive. Glutathione peroxidase and superoxide dismutase decreased during and after the dive (P < 0.05). Decreased leptin during the dive (P < 0.05) may have been related to the increased stress. Subjects had decreased energy intake and weight loss during the dive, similar to what is observed during spaceflight. Together, these similarities to spaceflight provide a model to use in further defining the physiologic effects of spaceflight and investigating potential countermeasures.