Corecovery of lipids and fermentable sugars from Rhodosporidium toruloides using ionic liquid cosolvents: Application of recycle to batch fermentation

This work evaluates the ability of an ionic liquid‐methanol cosolvent system to extract lipids and recycle fermentable sugars recovered from oil‐bearing Rhodosporidium toruloides grown in batch culture on defined media using glucose and xylose as carbon sources. Growth on the recycled mixed carbon substrate was successful with glucose consumed before xylose and overall cell mass to lipid yields (Y_P/X) between 57% and 61% (w/w relative to whole dried cell mass) achieved. Enzymatic hydrolysis of the delipified carbohydrate fraction recovered approximately 9%–11% (w/w) of the whole dried cell mass as fermentable sugars, which were successfully recycled as carbon sources without further purification. In total, up to 70% (w/w) of the whole dried cell mass was recovered as lipids and fermentable sugars and the substrate to lipid yields (Y_P/S) was increased from 0.12 to 0.16 g lipid/g carbohydrate consumed, highlighting the promise of this approach to process lipid bearing cell biomass. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1239–1242, 2014     

Domestication and the storage starch biosynthesis pathway: signatures of selection from a whole sorghum genome sequencing strategy

Next‐generation sequencing of complete genomes has given researchers unprecedented levels of information to study the multifaceted evolutionary changes that have shaped elite plant germplasm. In conjunction with population genetic analytical techniques and detailed online databases, we can more accurately capture the effects of domestication on entire biological pathways of agronomic importance. In this study, we explore the genetic diversity and signatures of selection in all predicted gene models of the storage starch synthesis pathway of Sorghum bicolor, utilizing a diversity panel containing lines categorized as either ‘Landraces’ or ‘Wild and Weedy’ genotypes. Amongst a total of 114 genes involved in starch synthesis, 71 had at least a single signal of purifying selection and 62 a signal of balancing selection and others a mix of both. This included key genes such as STARCH PHOSPHORYLASE 2 (SbPHO2, under balancing selection), PULLULANASE (SbPUL, under balancing selection) and ADP‐glucose pyrophosphorylases (SHRUNKEN2, SbSH2 under purifying selection). Effectively, many genes within the primary starch synthesis pathway had a clear reduction in nucleotide diversity between the Landraces and wild and weedy lines indicating that the ancestral effects of domestication are still clearly identifiable. There was evidence of the positional rate variation within the well‐characterized primary starch synthesis pathway of sorghum, particularly in the Landraces, whereby low evolutionary rates upstream and high rates downstream in the metabolic pathway were expected. This observation did not extend to the wild and weedy lines or the minor starch synthesis pathways.     

Conservative repair of a chromosomal double-strand break by single-strand DNA through two steps of annealing

The repair of chromosomal double-strand breaks (DSBs) is essential to normal cell growth, and homologous recombination is a universal process for DSB repair. We explored DSB repair mechanisms in the yeast Saccharomyces cerevisiae using single-strand oligonucleotides with homology to both sides of a DSB. Oligonucleotide-directed repair occurred exclusively via Rad52- and Rad59-mediated single-strand annealing (SSA). Even the SSA domain of human Rad52 provided partial complementation for a null rad52 mutation. The repair did not involve Rad51-driven strand invasion, and moreover the suppression of strand invasion increased repair with oligonucleotides. A DSB was shown to activate targeting by oligonucleotides homologous to only one side of the break at large distances (at least 20 kb) from the break in a strand-biased manner, suggesting extensive 5' to 3' resection, followed by the restoration of resected DNA to the double-strand state. We conclude that long resected chromosomal DSB ends are repaired by a single-strand DNA oligonucleotide through two rounds of annealing. The repair by single-strand DNA can be conservative and may allow for accurate restoration of chromosomal DNAs with closely spaced DSBs.     

Multifunctional grains for the future: genetic engineering for enhanced and novel cereal quality

Cereals provide more than half the world population’s calorific intake, and have a variety of other important uses as food and beverage ingredients, livestock feeds, and as sources of renewable energy and industrial components. The technology to genetically modify many important cereals is now well-established, thereby presenting new opportunities to produce cereals with enhanced quality and novel properties. In 2007, GM (genetically modified) maize with insect and herbicide resistance was grown on over 30 million hectares worldwide, yet to date, there are no GM cereals with enhanced or novel grain (end-use) qualities being grown in commercial farmers’ fields. This review will discuss some of the latest GM technology developments reported to enhance the quality of cereals for food and other uses. Developments and opportunities involving gene manipulation for starch and protein quality, as well as non-starch polysaccharides, phenolic compounds and micronutrients will also be discussed. The current paucity of GM cereals with enhanced grain quality is not related to the absence of technological progress, rather it is the regulatory and consumer acceptance issues that have slowed the release of these crops.     

IGFBP-3 Can Either Inhibit or Enhance EGF-mediated Growth of Breast Epithelial Cells Dependent upon the Presence of Fibronectin

Progression of breast cancer is associated with remodeling of the extracellular matrix, often involving a switch from estrogen dependence to a dependence on EGF receptor (EGFR)/HER-2 and is accompanied by increased expression of the main binding protein for insulin-like growth factors (IGFBP-3). We have examined the effects of IGFBP-3 on EGF responses of breast epithelial cells in the context of changes in the extracellular matrix. On plastic and laminin with MCF-10A normal breast epithelial cells, EGF and IGFBP-3 each increased cell growth and together produced a synergistic response, whereas with T47D breast cancer cells IGFBP-3 alone had no effect, but the ability of EGF to increase cell proliferation was markedly inhibited in the presence of IGFBP-3. In contrast on fibronectin with MCF-10A cells, IGFBP-3 alone inhibited cell growth and blocked EGF-induced proliferation. With the cancer cells, IGFBP-3 alone had no effect but enhanced the EGF-induced increase in cell growth. The insulin-like growth factor-independent effects of IGFBP-3 alone on cell proliferation were completely abrogated in the presence of an EGFR, tyrosine kinase inhibitor, Iressa. Although IGFBP-3 did not affect EGFR phosphorylation [Tyr¹⁰⁶⁸], it was found to modulate receptor internalization and was associated with activation of Rho and subsequent changes in MAPK phosphorylation. The levels of fibronectin and IGFBP-3 within breast tumors may determine their dependence on EGFR and their response to therapies targeting this receptor.     

The effect of an on-body personal lift assist device (PLAD) on fatigue during a repetitive lifting task

Occupations demanding frequent and heavy lifting are associated with an increased risk of injury. A personal lift assist device (PLAD) was designed to assist human muscles through the use of elastic elements. This study was designed to determine if the PLAD could reduce the level of general and local back muscle fatigue during a cyclical lifting task. Electromyography of two erector spinae sites (T9 and L3) was recorded during a 45-min lifting session at six lifts/lowers per minute in which male participants (n = 10) lifted a box scaled to represent 20% of their maximum back extensor strength. The PLAD device reduced the severity of muscular fatigue at both muscle sites. RMS amplitude increased minimally (22% and 26%) compared to the no-PLAD condition (104% and 88%). Minimal median frequency decreases (0.33% and 0.41%) were observed in the PLAD condition compared to drops of 12% and 20% in the no-PLAD condition. The PLAD had an additional benefit of minimizing pre–post changes in muscular strength and endurance. The PLAD also resulted in a significantly lower rate of perceived exertion across the lifting session. It was concluded that the PLAD was effective at decreasing the level of back muscular fatigue.     

Differential gene expression analysis by DNA microarrays technology and its application in molecular oncology

Accumulation of genetic and epigenetic aberrations leads to malignant transformation of normal cells. Functional studies of cancer using genomic and proteomic tools will help to reveal the true complexity of the processes leading to cancer development in humans. Until recently, diagnosis and prognosis of cancer was based on conventional pathologic criteria and epidemiological evidence. Certain tumors were divided only into relatively broad histological and morphological subcategories. Rapidly developing methods of differential gene expression analysis promote the search for clinically relevant genes changing their expression levels during malignant transformation. DNA microarrays offer a unique possibility to rapidly assess the global expression picture of thousands genes in any given time point and compare the detailed combinatory analysis results of global expression profiles for normal and malignant cells at various functional stages or separate experimental conditions. Acquisition of such "genetic portraits" allows searching for regularity and difference in expression patterns of certain genes, understanding their function and pathological importance, and ultimately developing the "molecular nosology" of cancer. This review describes the basis of DNA microarray technology and methodology, and focuses on their applications in molecular classification of tumors, drug sensitivity and resistance studies, and identification of biological markers of cancer.     

Ras/MAPK pathway confers basement membrane dependence upon endoderm differentiation of embryonic carcinoma cells

The formation of extraembryonic endoderm is one of the earliest steps in the differentiation of pluripotent cells of the inner cell mass during the early stages of embryonic development. The primitive endoderm cells and the derived parietal and visceral endoderm cells gain the capacity to produce collagen IV and laminin. The deposition of these components results in the formation of basement membrane and epithelium of the endoderm, with polarized cells covering the inner surface of the blastocoels. We used retinoic acid-induced endoderm differentiation of stem cell-like F9 embryonic carcinoma cells to study the role of the Ras pathway and its regulation in the formation of the visceral endoderm. Upon endoderm differentiation of F9 cells induced by retinoic acid, c-Fos expression, the downstream target of the Ras pathway, is suppressed by uncoupling Elk-1 phosphorylation/activation to MAPK activity. However, attachment to matrix gel greatly enhances the activation of MAPK in endoderm cells but not in undifferentiated F9 cells. Enhanced MAPK activation as a result of contact with basement membrane is able to compensate for reduced Elk-1 phosphorylation and c-Fos expression. We conclude that endoderm differentiation renders the activation of the Ras pathway basement membrane dependent, contributing to the epithelial organization of the visceral endoderm.