Simultaneous saccharification and fermentation of Kanlow switchgrass pretreated by hydrothermolysis using Kluyveromyces marxianus IMB4

A thermotolerant yeast strain named Kluyveromyces marxianus IMB4 was used in a simultaneous saccharification and fermentation (SSF) process using Kanlow switchgrass as a feedstock. Switchgrass was pretreated using hydrothermolysis at 200 degrees C for 10 min. After pretreatment, insoluble solids were separated from the liquid prehydrolyzate by filtration and washed with deionized water to remove soluble sugars and inhibitors. Insoluble solids were then hydrolyzed using a commercial cellulase preparation and the released glucose was fermented to ethanol by K. marxianus IMB4 in an SSF process. SSF temperature was 37, 41, or 45 degrees C and pH was 4.8 or 5.5. SSF was conducted for 7 days. Results were compared with a control of Saccharomyces cerevisiae D(5)A at 37 degrees C and pH 4.8. Fermentation by IMB4 at 45 and 41 degrees C ceased after 3 and 4 days, respectively, when a pH 4.8 citrate buffer was used. Fermentation continued for all 7 days using IMB4 at 37 degrees C and the control. When pH 5.5 citrate buffer was used, fermentation ceased after 96 h using IMB4 at 45 degrees C, and ethanol yield was greater than when pH 4.8 citrate buffer was used (78% theoretical). Ethanol yield using IMB4 at 45 degrees C, pH 5.5 was greater than the control after 48, 72, and 96 h (P < 0.05).     

The AUG start codon of the Saccharomyces cerevisiae NFS1 gene can be substituted for by UUG without increased initiation of translation at downstream codons.

The selection of the site for initiation of translation for the Saccharomyces cerevisiae NFS1 gene was examined using mutated AUG1, AUG2 and AUG3 codons. When AUG1 of the yeast NFS1 gene was mutated to UUG and the resulting mRNA was translated in vitro using a reticulocyte system, initiation from the mutated codon was abolished and occurred instead at downstream codons at increased rates. When the same mRNA was translated using a yeast extract, translation initiated at the mutated codon, albeit at a reduced rate, and there was no increased translation at downstream AUG codons. The NFS1 gene in which AUG1 was replaced by UUG was also able to substitute for the wild-type gene in vivo in yeast. Western blots confirmed that the encoded protein was the same size as that encoded by the wild-type gene and that both the wild-type and mutated proteins localized to mitochondria. This is apparently the first example of a yeast protein where mutagenesis of AUG1 does not lead to alternate use of a downstream AUG.     

Microaerobic hydrogen production by photosynthetic bacteria in a double-phase photobioreactor

The rate of hydrogen production by the marine nonsulfur photosynthetic bacterium, Rhodovulum sp., increased with increasing light intensity. A light intensity of 1800 W/m(2) hydrogen production rate was achieved at the rate of 9.4 micromol/mg dry weight/h. The hydrogen production of this strain was enhanced by the addition of a small amount of oxygen (12 micromol O(2)/reactor). Intracellular ATP content was most efficiently accumulated under microaerobic, dark conditions. Hydrogen production rate by Rhodovulum sp. was investigated using a double-phase photobioreactor consisting of light and dark compartments. This rate was compared with data obtained using a conventional photobioreactor. Rhodovulum sp. produced hydrogen at a rate of 0.38+/-0.03 micromol/mg dry weight/h under microaerobic conditions using the double-phase photobioreactor. The hydrogen production rate was four times greater under microaerobic conditions, as compared with anaerobic conditions using either type of photobioreactor. Hydrogen production using a double-phase photobioreactor was demonstrated continuously at the same rate for 150 h.     

The glass transition behavior of the globular protein bovine serum albumin

The glass-like transition behavior of concentrated aqueous solutions of bovine serum albumin was examined using rheological techniques. At mass fractions >0.4, there was a marked concentration dependence of viscosity with a glass-like kinetic arrest observed at mass fractions in the region of 0.55. At mass fractions >0.6 the material behaved as a solid with a Young's modulus rising from approximately 20 MPa at a mass fraction of 0.62-1.1 GPa at 0.86. The solid was viscoelastic and exhibited stress relaxation with relaxation times increasing from 33 to 610 s over the same concentration range. The concentration dependence of the osmotic pressure was measured, at intermediate concentrations, using an osmotic stress technique and could be described using a hard sphere model, indicating that the intermolecular interactions were predominantly repulsive. In summary, a major structural relaxation results from the collective motion of the globules at the supra-globule length scale and, at 20 degrees C, this is arrested at water contents of 40% w/w. This appears to be analogous to the glass transition in colloidal hard spheres.     

The effect of hyperthermia on transmembrane potential in Chinese hamster ovary cells in vitro

The effect of elevated temperature on transmembrane potential was studied in Chinese hamster ovary cells in vitro using tetraphenylphosphonium cation (TPP+) and 3,3'-dipentyloxacarbocyanine [Di-O-C5(3)], two unrelated lipophilic cation probes that equilibrate across the plasma membrane according to the transmembrane potential. Uptake of TPP+ was measured using a tritium-labeled probe and the uptake of the fluorescent probe Di-O-C5(3) was measured by flow cytometry. The Nernst equation was used to calculate transmembrane potential. The absolute values obtained for transmembrane potential at 37 degrees C using the two probes were different, but qualitatively similar results were obtained using either probe in the hyperthermia studies. Transmembrane potential measured at 43 and 45 degrees C was at least 20% higher than that measured at 37 degrees C, and the difference was statistically significant (P = 0.025 and P less than 0.01, respectively). The hyperpolarization induced by exposure to 45 degrees C persisted temporarily after cells had been returned to 37 degrees C. The hyperpolarization at 37 degrees C associated with a previous exposure to hyperthermia was maximal after cells had been held at 45 degrees C for 2.0 min, and fell to normal levels after 15.0 min at 37 degrees C.     

Coconut oil cake--a potential raw material for the production of alpha-amylase

Solid-state fermentation (SSF) was carried out using coconut oil cake (COC) as substrate for the production of alpha-amylase using a fungal culture of Aspergillus oryzae. Raw COC supported the growth of the culture, resulting in the production of 1372 U/gds alpha-amylase in 24 h. Process optimization using a single parameter mode showed enhanced enzyme titre, which was maximum (1827 U/gds) when SSF was carried out at 30 degrees C for 72 h using a substrate with 68% initial moisture. Supplementation with glucose and starch further enhanced enzyme titre, which was maximum (1911 U/gds) with 0.5% starch. However, maltose inhibited the enzyme production. Studies on the effect of addition of external organic and inorganic nitrogenous compounds further showed a positive impact on enzyme synthesis by the culture. Increase of 1.7-fold in the enzyme activity (3388 U/gds) was obtained when peptone at 1% concentration was added to the fermentation medium. The enzyme production was growth-related, the activity being the maximum when the fungal biomass was at its peak at 72 h. Use of COC as raw material for enzyme synthesis could be of great commercial significance. To the best of our knowledge this is the first report on alpha-amylase production using COC in SSF.     

Properties of protoplasts from the thermophile Bacillus coagulans and their significance for genetic studies

Regeneration of protoplasts of Bacillus coagulans was optimized by using low lysozyme concentrations and glycerol as the osmotic support. Protoplasts formed from cells grown at higher temperatures were thermostable and capable of regeneration at 55°C. Transfer of plasmids pAB224 and pUB110, using either whole cells or protoplast transformation was not achieved, despite using a variety of conditions. However, plasmid transfer was achieved by fusion with B. subtilis protoplasts containing plasmid pAB224.     

Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates

Stable isotope labeling is at present one of the most powerful methods in quantitative proteomics. Stable isotope labeling has been performed at both the protein as well as the peptide level using either metabolic or chemical labeling. Here, we present a straightforward and cost-effective triplex quantification method that is based on stable isotope dimethyl labeling at the peptide level. Herein, all proteolytic peptides are chemically labeled at their alpha- and epsilon-amino groups. We use three different isotopomers of formaldehyde to enable the parallel analysis of three different samples. These labels provide a minimum of 4 Da mass difference between peaks in the generated peptide triplets. The method was evaluated based on the quantitative analysis of a cell lysate, using a typical "shotgun" proteomics experiment. While peptide complexity was increased by introducing three labels, still more than 1300 proteins could be identified using 60 microg of starting material, whereby more than 600 proteins could be quantified using at least four peptides per protein. The triplex labeling was further utilized to distinguish specific from aspecific cAMP binding proteins in a chemical proteomics experiment using immobilized cAMP. Thereby, differences in abundance ratio of more than two orders of magnitude could be quantified.     

Enantioselective separation of phenylglycidates by capillary electrophoresis employing sulfated beta-cyclodextrin as chiral selector

A capillary electrophoresis (CE) method for the enantioseparation of phenylglycidates has been developed. Successful enantioseparation was achieved using sulfated beta-cyclodextrin as chiral selector in a phosphate buffer. The effects of varying pH, sulfated beta-cyclodextrin concentration and electrophoresis voltage were systematically investigated and the optimized separation conditions were thus obtained. When the migration time was set at the threshold value, it was found that the best enantioseparation was obtained at 10 kV with 3% (w/v) sulfated beta-cyclodextrin at pH 6.5. A range of substituted phenylglycidates were successfully separated using the method and the results shown to be superior to those obtained using gas chromatography (GC).     

Synthesis of S-alkyl and C-terminal analogs of the Saccharomyces cerevisiae a-factor. Influence of temperature on the stability of Fmoc and OFm groups toward HF

The a-mating factor of Saccharomyces cerevisiae Tyr-Ile-Ile-Lys-Gly-Val-Phe-Trp-Asp-Pro-Ala-Cys(farnesyl)OCH3, and 10 analogs modified at the cysteine side chain and/or the terminal carboxyl were synthesized using a combination of solid phase and solution phase methodologies. The strategy of synthesis involved the condensation of an amine terminal protected decapeptide with a carboxyl terminal S-alkylated dipeptide ester or amide using benzotriazol-l-yloxy-tris(methylamino)-phosphonium hexafluorophosphate as the coupling agent. The protected decapeptide was assembled on a PAM-resin using 9-fluorenylmethoxycarbonyl (Fmoc) for the protection of the Tyr alpha-amine and Lys epsilon-amine and 9-fluorenylmethyl ester (OFm) for the protection of the Asp beta-carboxyl. Premature loss of the OFm group from the HF cleavage was observed at 0-2 degrees, whereas no loss occurred when the cleavage reaction was conducted at -5 degrees. In contrast to these results, the OFm group in Asp(OFm) was partially removed by HF at -5 degrees and was completely stable to HF only at -20 degrees. The S-alkylated dipeptide esters were prepared, in yields from 64% to 88%, via thioalkylation of amine protected or unprotected dipeptide esters using potassium fluoride dihydrate as the base. The use of a tertiary amine as the base of thiohexadecanylation resulted in low reactivity.     

High-level production of recombinant chicken interferon-gamma by Brevibacillus choshinensis

Cytokines, such as interferon-gamma have been shown to have adjuvant and growth promoting activity in poultry and livestock and have the potential to be used as alternatives to antibiotics. We have developed an efficient system for commercial-scale synthesis of recombinant chicken interferon-gamma (ChIFN-gamma) using Brevibacillus choshinensis as the host for protein production. The ChIFN-gamma expression vector, pNCIFN, was constructed using the novel Escherichia coli-B. choshinensis shuttle vector, pNCMO2. ChIFN-gamma expression was optimized by investigating different culture conditions and different host B. choshinensis mutants. The highest level of production was observed using the B. choshinensis HPD31-MB2 strain grown at 30 degrees C, where ChIFN-gamma was produced at approximately 300-500 mg/L. ChIFN-gamma was also produced as a His-tagged fusion protein by using the pNCHis-IFN expression vector, a derivative of pNCMO2. The protein was constitutively secreted into the culture supernatant and could be partially purified in a single step using a Ni-nitrilotriacetic acid column. This recombinant His-ChIFN-gamma was shown to have the same biological activity as native ChIFN-gamma.     

Non-destructive analysis of the moisture content in shiitake mushrooms (Lentinula edodes) using near-infrared imaging at 1450 nm

Moisture content (MC) is an important factor in the freshness of shiitake mushrooms (Lentinula edodes). In this study, we developed a simple prediction method for MC of shiitake mushrooms using near-infrared (NIR) imaging at 1450 nm, as the band at 1450 nm is strongly correlated with OH groups. First, a NIR image was captured by an InGaAs sensor with a bandpass filter of 1450 nm; the captured image was then calibrated using an image taken at 1100 nm to remove shade and shape effects of mushrooms. The actual MC of shiitake mushrooms was measured using an oven-drying method, and a calibration curve was obtained for the cap surface and the gill side, respectively. The correlation coefficient reached more than 0.85, and the standard error of calibration ranged from 3.1% to 8.2%. Non-destructive MC images were quantified using the constructed calibration curves, and the experimental results showed that differences in the water-holding property can be visualized over 6 h for each side using our method.     

Interactions between carrageenans and milk proteins: a microstructural and rheological study

Gels were produced using kappa-, iota-, or hybrid-carrageenan at a low (0.2-0.25%) and a high (0.7-1.0%) dosage in skim milk. The microstructure of carrageenan and protein was observed by confocal laser scanning microscopy using direct immunostaining. Additionally, rheology was used to characterize the gels. The low kappa- and iota-carrageenan dosages resulted in gels with a fine stranded carrageenan-protein microstructure and emulsion-like inclusions, while the high dosages resulted in strongly flocculated microstructures. Hybrid-carrageenan exhibited flocculation at both dosages. When using iota- and hybrid-carrageenan at a high dosage and kappa-carrageenan at both dosages, the gel characteristics were dominated by carrageenan-carrageenan interactions. On the other hand, the gel with a low dosage of iota-carrageenan in milk was barely fusible, indicating the presence of a true coupled network. We suggest that kappa-, iota-, and hybrid-carrageenan all interact with casein micelles but that the impact of this interaction on the total gel properties varied.     

Effects of raloxifene on changes in bone density

Raloxifene hydrochloride therapy effectiveness in bone mineral density (BMD) changes compared to calcium and vitamin D3 therapy over a 2-year period. Case-control study: a group of 254 women was prescribed raloxifene (raloxifene hydrochloride) together with calcium and vitamin D3 while other group of 254 women used calcium and vitamin D3 therapy. BMD was measured at the hip, spine and forearm at the beginning and at the end of the 2-year period. Treatment with raloxifene resulted in a 3.7% increase in BMD at the spine in 98% of examinees. A 1.2% BMD increase was shown in 75% of examinees at the hip. A 1.2% decrease in BMD at forearm shown in 93% of examinees using raloxifene. The calcium and vitamin D3 therapy led to an increase in BMD in 58% examinees at the spine, in 56% at the hip and in 38% at the forearm, which was significantly lower than in women using raloxifene. Among women using calcium and vitamin D alone an average BMD decrease of 1.2% was registered on 42% of examinees at the spine, 2.6% decrease on 46% of examinees at the hip and 4.2% decrease on 35% of examinees at the forearm. Treatment with raloxifene resulted in a significant increase in BMD at the spine with odds ratio (OR 5.85, p <0.05) compared with calcium and vitamin D3 alone. There was no statistically proven increase in BMD at either the hip (OR 0.015) or forearm (OR 0.122).     

Tandem ligation at X-Cys and Gly-Gly positions via an orthogonally protected auxiliary group

4,5-dimethoxy-2-mercaptobenzylamine (Dmmb) has been protected by acetamidomethyl (Acm) and incorporated into a peptide thioester for use in tandem native chemical ligation. Upon ligation between the thioester and a Cys-peptide, Acm was removed from Dmmb using silver acetate, and a second ligation reaction was done at the Dmmb position. Dmmb removal using TFMSA-TFA effected overall tandem ligation at X-Cys and Gly-Gly.     

Design and analysis of orthogonally compliant features for local contact pressure relief in transtibial prostheses

A very attractive advantage of manufacturing prosthetic sockets using solid freeform fabrication is the freedom to introduce design solutions that would be difficult to implement using traditional manufacturing techniques. Such is the case with compliant features embedded in amputee prosthetic sockets to relieve contact pressure at the residual limb-socket interface. The purpose of this study was to present a framework for designing compliant features to be incorporated into transtibial sockets and manufacturing prototypes using selective laser sintering (SLS) and Duraform material. The design process included identifying optimal compliant features using topology optimization algorithms and integrating these features within the geometry of the socket model. Using this process, a compliant feature consisting of spiral beams and a supporting external structure was identified. To assess its effectiveness in reducing residual limb-socket interface pressure, a case study was conducted using SLS manufactured prototypes to quantify the difference in interface pressure while a patient walked at his self-selected pace with one noncompliant and two different compliant sockets. The pressure measurements were performed using thin pressure transducers located at the distal tibia and fibula head. The measurements revealed that the socket with the greatest compliance reduced the average and peak pressure by 22% and 45% at the anterior side distal tibia, respectively, and 19% and 23% at the lateral side of the fibula head, respectively. These results indicate that the integration of compliant features within the socket structure is an effective way to reduce potentially harmful contact pressure and increase patient comfort.     

Capture of a monoclonal antibody and prediction of separation conditions using a synthetic multimodal ligand attached on chips and beads

A synthetic ligand called 2-mercapto-5-benzimidazolesulfonic acid has been successfully used for the specific chromatographic capture of antibodies from a cell culture supernatant. Adsorption occurred at physiological ionic strength and pH range between 5.0 and 6.0, with some binding capacity variations within this pH range: antibody uptake increased when the pH decreased. With very dilute feedstocks, as was the case with the cell culture supernatant under investigation, it was found that the pH had to be slightly lowered to get a good antibody sorption capacity. To optimize separation conditions, a preliminary study was made using ProteinChip Arrays that displayed the same chemical functionalities as the resin. Arrays were analyzed using SELDI-MS. By this mean, it was possible to cross-over simultaneously different pH conditions at the adsorption and the desorption steps. Best conditions were implemented for preparative separation using regular lab-scale columns. At pH 5.2, antibody adsorption was not complete, while at pH 5.0 the antibody was entirely captured. pH 9 was selected at elution, rather than pH 8.0 or 10.0, and resulted in a complete desorption of antibodies from the column. Benefits of the prediction of separation conditions of antibodies on MBI beads using SELDI-MS were a significant reduction in analysis time and in sample volume. This was possible because the separation of IgG on the chip surface did mimic very well the separation on beads.     

A polyhydroxyalkanoates bioprocess improvement case study based on four fed-batch feeding strategies

The modelling and optimization of a process for the production of the medium chain length polyhydroxyalkanoate (mcl-PHA) by the bacterium Pseudomonas putida KT2440 when fed a synthetic fatty acid mixture (SFAM) was investigated. Four novel feeding strategies were developed and tested using a constructed model and the optimum one implemented in further experiments. This strategy yielded a cell dry weight of 70.6 g l⁻¹ in 25 h containing 38% PHA using SFAM at 5 l scale. A phosphate starvation strategy was implemented to improve PHA content, and this yielded 94.1 g l⁻¹ in 25 h containing 56% PHA using SFAM at 5 l scale. The process was successfully operated at 20 l resulting in a cell dry weight of 91.2 g l⁻¹ containing 65% PHA at the end of a 25-h incubation.     

Development of Kudoa thyrsites (Myxozoa: Myxosporea) in netpen-reared Atlantic salmon determined by light microscopy and a polymerase chain reaction test.

The intramuscular phase of development of Kudoa thyrsites, the myxosporean associated with post-mortem myoliquefaction, or 'soft flesh syndrome', is described using histological preparations of the musculature of seawater netpen-reared Atlantic salmon Salmo salar. Hatchery-reared Atlantic salmon were naturally exposed to the infective stage while held in the experimental seawater netpens of the Pacific Biological Station in Nanaimo, British Columbia, Canada. In fish exposed during the summer of 1995, K. thyrsites infections were first detected in the somatic musculature at 13 wk post-exposure (p.e.) using only light microscopy. In the 1997 exposure, infections were first detected at 6 wk p.e. using a PCR test and at 9 wk p.e. using light microscopy. The earliest stage detected by histology was a small plasmodium containing 4 nuclei. No host response was observed that was directly related to the presence of intact plasmodia within muscle fibers. However, a response was associated with ruptured plasmodia, which was characterized by chronic, multifocal inflammation between the muscle fibers.     

Gastric Pepsin in an Anuran Larva

Larval stomach development was studied in the obligate carnivorous larva of the frog Lepidobatrachus laevis . Pepsin producing cells of the larval stomach were identified using rabbit anti-porcine pepsin and immunohistochemical techniques. Pepsin production was detected at a very early stage of development (stage 24: during opercular development) when the larvae were first competent for feeding. Peptic activity in isolated larval stomachs was demonstrated in a microassay using acid denatured hemoglobin at pH 1.7. The total activity per stomach increased 5,400 fold through the beginning of metamorphosis and the specific activity increased 345 fold through the same period. Electrophoretic analysis of the larval pepsinogens, using a caseinolytic assay revealed the presence of one major pepsinogen at stage 24; two additional isozymes were observed during later larval development. The molecular weight of the isopepsinogens was 34,800.