MORPHOLOGICAL CHANGES AND THE REQUIREMENTS FOR MACROMOLECULE SYNTHESIS DURING EXCYSTMENT OF ACANTHAMOEBA CASTELLANII

Light and phase-contrast microscopic observations of excystment in Acanthamoeba castellanii have been used to classify cells in excysting populations as free trophozoites, or mature, activated, or preemergent cysts. These categories have been used to describe the kinetics of excystment. A pH of 7 and a temperature of 30°C have been found to be optimal for the activation of mature cysts. Both activation and emergence are inhibited by cycloheximide and actinomycin D, but neither process is much affected by hydroxyurea. Cell-free extracts of high molecular weight components of cyst cytoplasm can support protein synthesis in vitro, although less efficiently than similar extracts from trophozoites. Evidence indicates that some of the functional RNA in the cyst extracts is synthesized before excystment.     

The dependency of fetal left ventricular biomechanics function on myocardium helix angle configuration

The helix angle configuration of the myocardium is understood to contribute to the heart function, as finite element (FE) modeling of postnatal hearts showed that altered configurations affected cardiac function and biomechanics. However, similar investigations have not been done on the fetal heart. To address this, we performed image-based FE simulations of fetal left ventricles (LV) over a range of helix angle configurations, assuming a linear variation of helix angles from epicardium to endocardium. Results showed that helix angles have substantial influence on peak myofiber stress, cardiac stroke work, myocardial deformational burden, and spatial variability of myocardial strain. A good match between LV myocardial strains from FE simulations to those measured from 4D fetal echo images could only be obtained if the transmural variation of helix angle was generally between 110 and 130°, suggesting that this was the physiological range. Experimentally discovered helix angle configurations from the literature were found to produce high peak myofiber stress, high cardiac stroke work, and a low myocardial deformational burden, but did not coincide with configurations that would optimize these characteristics. This may suggest that the fetal development of myocyte orientations depends concurrently on several factors rather than a single factor. We further found that the shape, rather than the size of the LV, determined the manner at which helix angles influenced these characteristics, as this influence changed significantly when the LV shape was varied, but not when a heart was scaled from fetal to adult size while retaining the same shape. This may suggest that biomechanical optimality would be affected during diseases that altered the geometric shape of the LV.

     

Motor learning by observing

Learning complex motor behaviors like riding a bicycle or swinging a golf club is based on acquiring neural representations of the mechanical requirements of movement (e.g., coordinating muscle forces to control the club). Here we provide evidence that mechanisms matching observation and action facilitate motor learning. Subjects who observed a video depicting another person learning to reach in a novel mechanical environment (imposed by a robot arm) performed better when later tested in the same environment than subjects who observed similar movements but no learning; moreover, subjects who observed learning of a different environment performed worse. We show that this effect is not based on conscious strategies but instead depends on the implicit engagement of neural systems for movement planning and control.     

Long-Term Reproducible Expression in Human Fetal Liver Hematopoietic Stem Cells with a UCOE-Based Lentiviral Vector

Hematopoietic Stem Cell (HSC) targeted gene transfer is an attractive treatment option for a number of hematopoietic disorders caused by single gene defects. However, extensive methylation of promoter sequences results in silencing of therapeutic gene expression. The choice of an appropriate promoter is therefore crucial for reproducible, stable and long-term transgene expression in clinical gene therapy. Recent studies suggest efficient and stable expression of transgenes from the ubiquitous chromatin opening element (UCOE) derived from the human HNRPA2B1-CBX3 locus can be achieved in murine HSC. Here, we compared the use of HNRPA2B1-CBX3 UCOE (A2UCOE)-mediated transgene regulation to two other frequently used promoters namely EF1α and PGK in human fetal liver-derived HSC (hflHSC). Efficient transduction of hflHSC with a lentiviral vector containing an HNRPA2B1-CBX3 UCOE-eGFP (A2UCOE-eGFP) cassette was achieved at higher levels than that obtained with umbilical cord blood derived HSC (3.1x; p<0.001). While hflHSC were readily transduced with all three test vectors (A2UCOE-eGFP, PGK-eGFP and EF1α-eGFP), only the A2-UCOE construct demonstrated sustained transgene expression in vitro over 24 days (p<0.001). In contrast, within 10 days in culture a rapid decline in transgene expression in both PGK-eGFP and EF1α-eGFP transduced hflHSC was seen. Subsequently, injection of transduced cells into immunodeficient mice (NOD/SCID/Il2rg ^-/-) demonstrated sustained eGFP expression for the A2UCOE-eGFP group up to 10 months post transplantation whereas PGK-eGFP and EF1α-eGFP transduced hflHSC showed a 5.1 and 22.2 fold reduction respectively over the same time period. We conclude that the A2UCOE allows a more efficient and stable expression in hflHSC to be achieved than either the PGK or EF1α promoters and at lower vector copy number per cell.     

Regionally-Specified Second Trimester Fetal Neural Stem Cells Reveals Differential Neurogenic Programming

Neural stem/progenitor cells (NSC) have the potential for treatment of a wide range of neurological diseases such as Parkinson Disease and multiple sclerosis. Currently, NSC have been isolated only from hippocampus and subventricular zone (SVZ) of the adult brain. It is not known whether NSC can be found in all parts of the developing mid-trimester central nervous system (CNS) when the brain undergoes massive transformation and growth. Multipotent NSC from the mid-trimester cerebra, thalamus, SVZ, hippocampus, thalamus, cerebellum, brain stem and spinal cord can be derived and propagated as clonal neurospheres with increasing frequencies with increasing gestations. These NSC can undergo multi-lineage differentiation both in vitro and in vivo, and engraft in a developmental murine model. Regionally-derived NSC are phenotypically distinct, with hippocampal NSC having a significantly higher neurogenic potential (53.6%) over other sources (range of 0%–27.5%, p<0.004). Whole genome expression analysis showed differential gene expression between these regionally-derived NSC, which involved the Notch, epidermal growth factor as well as interleukin pathways. We have shown the presence of phenotypically-distinct regionally-derived NSC from the mid-trimester CNS, which may reflect the ontological differences occurring within the CNS. Aside from informing on the role of such cells during fetal growth, they may be useful for different cellular therapy applications.     

Alpha-toxin production by Clostridium septicum at different culture conditions

Clostridium septicum is responsible for a number of diseases, the most serious of which are a frequently fatal non-traumatic gas gangrene in man and braxy malignant oedema and blackquarter in farm animals. Immunity to these diseases is mediated mainly by antitoxin raised against C. septicum alpha toxin, which has hemolytic, lethal and necrotizing activities.Clostridium septicum produces lethal antigen only in low titre and in a variable way. In addition, the immunogenicity of native toxic filtrates is weak, which results in poor antibody response in animals. The aim of this work was to determine the best culture conditions for obtention of the protective antigen. To do this, the presence of the alpha toxin was identified in culture filtrates by means of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under different culture conditions. The strain used was C. septicum 3606. The cultures were performed in anaerobic conditions at 37 degrees C. Two systems were employed: (a) batch (0.5% initial glucose concentration); and (b) batch with partial feeding of the carbon source (0.1% glucose concentration) at controlled pH. The hemolytic activity of supernatants was determined using human erythrocytes, and the final biomass concentration was estimated by dry weight determination. The proteins present in supernatants were concentrated by ultrafiltration and identified in 10% SDS-polyacrylamide gels. With partial feeding of the carbon source the final biomass (2.22 g/L) was three times higher than the amount reached in batch system (0.70 g/L), however, no difference was found in the hemolytic activity (16 HU50/mL) showing no correlations between cell growth and hemolytic activity of the supernatants. Electrophoresis of filtrate cultures obtained with partial feeding of the carbon source allowed us to identify several bands, two of them corresponded to non-active alpha-protoxin (46 kDa) and the active toxin (42 kDa) and the aggregate of the active toxin at the seeding line. On the other hand, the ultrafiltrate from batch system showed less number of bands and the 46 kDa band was much weaker, suggesting a lower toxin production in this system. It becomes important to determine culture conditions and correlate with extracellular protein presence to optimize C. septicum antigen protector production.