Process design and optimization of novel wheat-based continuous bioethanol production system

A novel design of a wheat-based biorefinery for bioethanol production, including wheat milling, gluten extraction as byproduct, fungal submerged fermentation for enzyme production, starch hydrolysis, fungal biomass autolysis for nutrient regeneration, yeast fermentation with recycling integrated with a pervaporation membrane for ethanol concentration, and fuel-grade ethanol purification by pressure swing distillation (PSD), was optimized in continuous mode using the equation-based software General Algebraic Modelling System (GAMS). The novel wheat biorefining strategy could result in a production cost within the range of dollars 0.96-0.50 gal(-1) ethanol (dollars 0.25-0.13 L(-1) ethanol) when the production capacity of the plant is within the range of 10-33.5 million gal y(-1) (37.85-126.8 million L y(-1)). The production of value-added byproducts (e.g., bran-rich pearlings, gluten, pure yeast cells) was identified as a crucial factor for improving the economics of fuel ethanol production from wheat. Integration of yeast fermentation with pervaporation membrane could result in the concentration of ethanol in the fermentation outlet stream (up to 40 mol %). The application of a PSD system that consisted of a low-pressure and a high-pressure column and employing heat integration between the high- and low-pressure columns resulted in reduced operating cost (up to 44%) for fuel-grade ethanol production.     

In vitro and in vivo assessment of vitamin A encapsulation in a liposome–protein delivery system

Vitamin A (VA) is an essential nutrient needed in small amounts by humans and supports a wide range of biological actions. Retinol, the most common and most biologically active form of VA has also been found to inhibit peroxidation processes in membranes and it has been widely used as an ingredient with pharmaceutical and nutritional applications. VA is a lipophilic molecule, sensitive to air, oxidizing agents, ultraviolet light and low pH levels. For these reasons, it is necessary for VA to be protected against oxidation. Another disadvantage in the application of VA is its low solubility in aqueous media. Both issues (sensitivity and solubility) can be solved by employing encapsulation techniques. Liposomes can efficiently encapsulate lipid-soluble materials, such as VA. The encapsulated materials are protected from environmental and chemical changes. A new liposome/β-lactoglobulin formulation has been developed as a stable delivery system for VA. The aim of this study was the encapsulation of VA into β-lactoglobulin–liposome complexes, recently developed in our laboratory. The in vivo bioavailability characterization of VA was tested after administration in laboratory animals (mice). In this report, we demonstrate that VA could be efficiently entrapped and delivered in a phospholipid–sterol–protein membrane resembling system, a newly synthesized promising carrier. Based on this finding, the phospholipid–sterol–protein membrane resembling system may be one of the promising approaches to enhance VA absorption and to overcome the formulation difficulties associated with lipophilic means. The carrier system described here has huge potential in food fortification applications to treat VA deficiency.     

Lateral flagella of Aeromonas species are essential for epithelial cell adherence and biofilm formation

Mesophilic Aeromonas strains express a single polar flagellum in all culture conditions and produce lateral flagella on solid media. Such hyperflagellated cells demonstrate increased adherence. Nine lateral flagella genes, lafA-U for Aeromonas hydrophila, and four Aeromonas caviae genes, lafA1, lafA2, lafB and fliU, were isolated. Mutant characterization, nucleotide and N-terminal sequencing demonstrated that the A. hydrophila and A. caviae lateral flagellins were almost identical, but were distinct from their polar flagellum counterparts. The aeromonad lateral flagellins exhibited higher molecular masses on SDS-PAGE, and this aberrant migration was thought to result from post-translational modification through glycosylation. Mutation of the Aeromonas lafB, lafS or both A. caviae lateral flagellins caused the loss of lateral flagella and a reduction in adherence and biofilm formation. Mutations in lafA1, lafA2, fliU or lafT resulted in strains that expressed lateral flagella, but had reduced adherence levels. Mutation of the lateral flagella loci did not affect polar flagellum synthesis, but the polarity of the transposon insertions on the A. hydrophila lafTlU genes resulted in non-motility. However, mutations that abolished polar flagellum production also inhibited lateral flagella expression. We conclude that Aeromonas lateral flagella: (i) play a role in adherence and biofilm formation; (ii) are distinct from the polar flagellum; (iii) synthesis is dependent upon the presence of a polar flagellum filament; and (iv) that the motor proteins of the polar and lateral flagella systems appear to be shared.     

Optimization of in vitro bovine embryo production: effect of duration of maturation,length of gamete co-incubation,sperm concentration and sire

The aim of these experiments was to investigate the effect of duration of IVM, duration of gamete co-incubation, and of sperm dose on the development of bovine embryos in vitro. In addition, the speed of sperm penetration of six bulls of known differing in vivo and in vitro fertility was examined. In Experiment 1, following IVM for 16, 20, 24, 28 or 32 h, cumulus oocyte complexes (COCs) were inseminated with 1 x 10(6) spermatozoa/ml. After 24 h co-incubation, presumptive zygotes were denuded and placed in droplets of synthetic oviduct fluid (SOF). In Experiment 2, following IVM and IVF, presumptive zygotes were removed from fertilization wells at 1, 5, 10, 15 or 20 h post insemination and placed in culture as described above. In Experiment 3, following IVM, COCs were inseminated with sperm doses ranging from 0.01 x 10(6) to 1 x 10(6) spermatozoa/ml. Following co-incubation for 24 h, presumptive zygotes were placed in culture as described above. In Experiment 4, following IVM, oocytes were inseminated with sperm from six bulls of known differing field fertility. To assess the rate of sperm penetration, oocytes were subsequently fixed every 3 h (up to 18 h) following IVF. Based on the results of Experiment 4, in Experiment 5, following IVM for 12, 18 or 24 h, COCs were inseminated with sperm from two sires with markedly different penetration speeds. After 24 h co-incubation, presumptive zygotes were denuded and placed in culture. The main findings from this study are that (1) the optimal duration of maturation of bovine oocytes in vitro to maximize blastocyst yield is 24 h, (2) sperm-oocyte co-incubation for 10 h is sufficient to ensure maximal blastocyst yields, (3) sperm concentrations of 0.25 x 10(6) and 0.5 x 10(6) spermatozoa/ml yielded significantly more blastocysts than any other concentration within the range of 0.01 x 10(6) 1 x 10(6) spermatozoa/ml, (4) there are marked differences in the kinetics of sperm penetration between sires and this may be a useful predictor of field fertility, and (5) the inferior development associated with slower penetration rates may in part be overcome by carrying out IVF at a time when the actual penetration is most likely to coincide with the completion of maturation.     

Mutations in the INK4a/ARF melanoma susceptibility locus functionally impair p14ARF

The INK4a/ARF locus encodes two cell cycle regulatory proteins, the cyclin-dependent kinase inhibitor, p16(INK4a), and the p53 activator, p14(ARF). Germline mutations in this locus are associated with melanoma susceptibility in 20-40% of multiple case melanoma families. Many of these mutations specifically impair p16(INK4a), whereas mutations uniquely targeting p14(ARF) are rare. Nevertheless, the importance of p14(ARF) has not been excluded because more than 40% of INK4a/ARF alterations affect p16(INK4a) and p14(ARF). We now report that p14(ARF) is functionally impaired in melanoma kindreds carrying INK4a/ARF mutations. Of the seven INK4a/ARF mutations tested, three altered the subcellular distribution of p14(ARF) and diminished the ability of p14(ARF) to activate the p53 pathway. This work establishes the importance of p14(ARF) in melanoma predisposition.     

Physical mapping of the human T-cell receptor beta gene complex,using yeast artificial chromosomes

Yeast artificial chromosomes (YACs) were used to construct a physical map of the germline human T-cell chain gene complex (TCRB). Variable region genes (BV) for the 25 known subfamilies were used as probes to screen the ICRF AM4x YAC library. Of the five positive YACs identified, one YAC designated B3, 820 kilobase pairs (kbp) in size, scored positive for all 25 TCRBV subfamilies plus the constant region genes (BC) when analyzed by pulse field gel electrophoresis. Restriction enzyme mapping of B3 located TCRBV and TCRBC gene regions to 4 Sfi I fragments of 280 110, 90, and 125 kbp and was in accordance with published data. In addition comparison of hybridization results of Sfi I-restricted B3 and genomic DNA from the parental cell line GM1416B revealed identical banding patterns. The data thus showed YAC B3 encoded a complete and unrearranged TCRB gene locus of some 600–620 kbp. The map was further resolved by locating restriction sites for Sal I and Bss HII on B3, giving more precise localization of the individual TCRBV gene families. Flourescent in situ hybridization of B3 to spreads of human metaphase chromosomes localized B3 to 7q35. However, two additional signals were obtained; one attributable to the TCRBV orphon cluster on 9p21, the second to the long arm of chromosome 2. Polymerase chain reaction amplification of a chromosome 2 somatic cell hybrid, using primers for all 25 TCRBV gene families, revealed that the signal was not attributable to a second orphon cluster. It is suggested that B3 is a chimeric YAC with an intact TCRB locus flanked by chromosome 2 sequences.