Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis

Background

In the last years, the biotechnological production of platform chemicals for fuel components has become a major focus of interest. Although ligno-cellulosic material is considered as suitable feedstock, the almost inevitable pretreatment of this recalcitrant material may interfere with the subsequent fermentation steps. In this study, the fungus Ustilago maydis was used to produce itaconic acid as platform chemical for the synthesis of potential biofuels such as 3-methyltetrahydrofuran. No studies, however, have investigated how pretreatment of ligno-cellulosic biomass precisely influences the subsequent fermentation by U. maydis. Thus, this current study aims to first characterize U. maydis in shake flasks and then to evaluate the influence of three exemplary pretreatment methods on the cultivation and itaconic acid production of this fungus. Cellulose enzymatically hydrolysed in seawater and salt-assisted organic-acid catalysed cellulose were investigated as substrates. Lastly, hydrolysed hemicellulose from fractionated beech wood was applied as substrate.

Results

U. maydis was characterized on shake flask level regarding its itaconic acid production on glucose. Nitrogen limitation was shown to be a crucial condition for the production of itaconic acid. For itaconic acid concentrations above 25 g/L, a significant product inhibition was observed. Performing experiments that simulated influences of possible pretreatment methods, U. maydis was only slightly affected by high osmolarities up to 3.5 osmol/L as well as of 0.1 M oxalic acid. The production of itaconic acid was achieved on pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction of pretreated beech wood.

Conclusion

The fungus U. maydis is a promising producer of itaconic acid, since it grows as single cells (yeast-like) in submerged cultivations and it is extremely robust in high osmotic media and real seawater. Moreover, U. maydis can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this fungus combines important advantages of yeasts and filamentous fungi. Nevertheless, the biomass pretreatment does indeed affect the subsequent itaconic acid production. Although U. maydis is insusceptible to most possible impurities from pretreatment, high amounts of salts or residues of organic acids can slow microbial growth and decrease the production. Consequently, the pretreatment step needs to fit the prerequisites defined by the actual microorganisms applied for fermentation.     

Amniocentesis and antenatal sex determination in the rhesus monkey (Macaca mulatta).

Antenatal sex was correctly identified in 87.5% of the rhesus monkeys studied by evaluation of sex chromatin bodies in the exfoliated cells present in amniotic fluid. Fluid samples were obtained by either amniocentesis, amnioscopy, or by cesarean section at 80--120 days of gestation. Amniotic fluid concentrations of progesterone, total estrogen, and testosterone were correlated with fetal sex. Concentrations of these hormones decreased over the time period examined. Amniocentesis is a simple procedure which provides for routine fetal sex determination.     

Embryotoxicity studies of norfloxacin in cynomolgus monkeys. II. Role of progesterone.

Norfloxacin, an orally active fluoroquinolone antimicrobial, has been reported to be embryolethal but not teratogenic when administered to pregnant cynomolgus macaques prior to gestational day (GD) 36 at doses > or = 200 mg/kg/day. Additional studies have been performed in an effort to examine the mechanism responsible for this effect, particularly regarding the role of progesterone (P). The first study (Study I) investigated the effect of norfloxacin administration during early pregnancy (200 mg/kg/day; daily GD 20-30) in the absence of a functional corpus luteum (CL). The CL was surgically removed from 16 gravid females on GD 19 in order to focus on placental-derived P; ten were dosed with norfloxacin and six received vehicle only. Embryolethality was observed for 7/10 (70%) of the treated animals during GD 25-31 versus 0/6 (0%) for controls. A reduction in serum P was noted prior to embryonic loss, although no significant effects on chorionic gonadotropin (CG), 17 beta-estradiol (E2), or P or E urinary metabolites were observed. A second study (Study II) was performed in order to evaluate the capacity of norfloxacin (200 mg/kg) to reduce CL-derived P in both normally cycling and CG-stimulated nonpregnant females (ten treated, ten controls; daily for 8 days). No effects on P production or on luteal phase or menstrual cycle lengths were observed. The third study (Study III) was designed to examine the effect of norfloxacin on the metabolism and excretion of P in nonpregnant females. Silastic P implants were placed subcutaneously in order to maintain constant P levels during a 10 day treatment regimen (200 mg/kg/day; ten controls, nine treated). Five of the controls and four of the norfloxacin-treated females also received 14C-P intravenously within 1 hr of the last dose of norfloxacin in order to study excretory patterns. No significant differences between control and treated groups were observed. The results of these studies combined suggest that the developmental toxic effects observed in prior studies and Study I are specific to pregnancy and directly related to placental-derived P production.     

Frequency of spontaneous congenital defects in rhesus and cynomolgus macaques

Abstract: This paper summarizes the spontaneous incidence of congenital defects in the rhesus and cynomolgus macaque colonies (Macaca mulatta and M. fascicularis) at the California Regional Primate Research Center. The computerized database used in this analysis included fetuses, term infants, juveniles, and adults that underwent a necropsy procedure over a 14-year period (1983–1996). The calculated malformation rates were 0.9% (40/4,390) and 0.3% (3/965) for the rhesus and cynomolgus monkey, respectively. Most of the observed malformations in both species affected the musculoskeletal and the cardiovascular systems, while a smaller number of defects were observed in the gastrointestinal, urogenital, endocrine, and central nervous systems. Inbreeding did not contribute to the spontaneous malformation incidence and there was no predilection for sex (male vs. female) or housing (indoors vs. outdoors) among the malformed cases. This spontaneous malformation database in our macaque colony aids in the interpretation of defects that occur in an experimental study as well as in the ongoing assessment of a healthy nonhuman primate breeding colony.     

The use of a urinary estrone conjugates assay for detection of optimal mating time in the cynomolgus macaque (Macaca fascicularis)

Forty-four female cynomolgus macaques (Macaca fascicularis) were examined to determine the optimum fertile period for mating. Daily urinary estrone conjugates (E1C) were measured, beginning on day 7 of the menstrual cycle, until a 1.5-gold E1C rise above the baseline was detected. The females were bred the next morning. Pregnancies were verified in all animals at day 18 postbreeding, and/or on day 25 postbreeding. Serum progesterone levels were used to correlate the relationship between ovulation and the E1C peak. Forty-four of the 57 cycles indicated a urinary E1C peak between days 10-15 of the menstrual cycle; this peak occurred on the day following the initial 1.5-fold to twofold rise in 90% of the cycles. A single 2-hr mating period the day before, the day of, or the day after the E1C peak resulted in conception in 17 of 44 (38.6%) animals.     

Effects of the oxygen minimum zone on squat lobster distributions in the Gulf of California, Mexico

Distribution of squat lobsters of the genera Gastropthychus (one species), Uroptychus (1), Janetogalathea (1), Galacantha (1), Munidopsis (8), and Munida (7) in the Gulf of California, Mexico, was plotted vs. the localization of the Oxygen Minimum Zone (OMZ) using bottom projections of the 0.50, 0.25, 0.10, and 0.05 ml l^?1 oxylines. Four fringes where oxygen concentrations were equal or lower than these concentrations were obtained, extending from the southern Gulf to about 28°–28°30′N. A total of 172 sampling locations with squat lobsters were plotted on four maps, one for each fringe, noting that with decreasing values of bottom oxygen (i.e., from 0.50 to 0.05 ml l^?1) the number of locations with species included between the upper and lower boundaries of these fringes decreases as follows: 34% for 0.50, 25% for 0.25, 12% for 0.10, and 5% for 0.05 ml l^?1. The upper and lower limits of the 0.05 ml l^?1 fringe are recognised as threshold frontiers, acting as barriers between shallow and deep communities of squat lobsters in the area. The distribution of the localities where species have been collected with respect to the upper and lower boundaries of the 0.05 ml l^?1 fringe clearly indicates a segregation pattern. Eight species of Munida (except M. perlata), and one each of Janetogalathea and Gastropthychus inhabit the shallow area, just above the OMZ core, with species of Munida generally found a rather long distance from this fringe. By contrast, species of Munidopsis, G. diomedeae and U. pubescens correspond to the deep area, i.e., below the OMZ core. It is suggested that the core of the OMZ in the Gulf of California serves as a vertical and latitudinal distribution filter, preventing species from freely migrating up and down or south and north.     

A new, easy, and rapid high-throughput detection method for the common GJB2 (CX26), 35delG mutation

GJB2 (Gap Junction protein beta type 2; Connexin 26, CX26) is known for its contribution to nonsyndromic recessive deafness (NSRD). One particular mutation, 35delG, a deletion of one guanine from a stretch of six leading to a frame shift early in the gene, has a high prevalence in populations from European descent. 35delG testing therefore has become a standard test in genetic diagnostic laboratories. Most of the currently available methods for the detection of 35delG are relatively time consuming, and not suited for high-throughput diagnostic testing. Within this paper we present a real-time PCR genotyping assay based on melting curve analysis, requiring only a single preparation step before the actual analysis. The assay was optimized on a panel of 48 samples with known 35delG genotypes and subsequently tested using a large Belgian population (N = 460) with unknown 35delG status. For the latter set of samples, real-time PCR results were validated with SNAPShot, an assay used in our laboratory for diagnostic purposes. The real-time PCR genotyping method has proven to be highly reliable, rapid, cost-effective, and suitable for high-throughput screening. We believe that this genetic test for 35delG will find widespread applications in the DNA diagnostic field.