Extended periods of neural induction and propagation of embryonic stem cell-derived neural progenitors with EGF and FGF2 enhances Lmx1a expression and neurogenic potential

Neural stem (NS) cells are multipotent cells defined by their capacity to proliferate and differentiate into all neuronal and glial phenotypes. NS cells can be obtained from specific regions of the adult brain, or generated from embryonic stem cells (ESCs). NS cells differentiate into neural progenitor (NP) cells and subsequently neural precursors, as transient steps towards terminal differentiation into specific mature neuronal or glial phenotypes. When cultured in EGF and FGF2, ESC-derived NS cells have been reported to be stable and multipotent. Conditions that enable differentiation of NS cells through the committed progenitor and precursor stages to specific neuronal subtypes have not been fully established. In this study we investigated, using Lmx1a reporter ESCs, whether the length of neural induction (NI) dictated the phenotypic potential of cultures of ESC-derived NS cells or NP cells. Following 4, 7 or 10 day periods of NI, ESCs in monolayer culture were harvested and cultured as neurospheres, prior to replating as monolayer cultures for several passages in EGF and FGF2. The NS/NP cultures were then directed towards mature neuronal fates over 16-17 days. 4 and 7-day NS cell cultures could not be differentiated towards dopaminergic, serotonergic or cholinergic fates as determined by the absence of tyrosine hydroxylase, 5-HT or choline acetyltransferase (ChAT) immunolabelling. In contrast NS/NP cultures derived after 10 days of NI were able to generate tyrosine hydroxylase and 5-HT positive neurons (24 ± 6 and 13 ± 1% of the βIII-tubulin positive population, respectively, n = 3). Our data suggest that extended periods of neural induction enhanced the potential of mouse ESC-derived NS/NP cells to generate specific subtypes of neurons. NS/NP cells derived after shorter periods of NI appeared to be lineage-restricted in relation to the neuronal subtypes observed after removal of EGF.     

Following cell-fate in E. coli after infection by phage lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant). We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells. Here, we describe the full procedure for performing the infection experiments described in our earlier work. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35°C to trigger viral DNA injection. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.     

Identification and characterization of NARROW AND ROLLED LEAF 1, a novel gene regulating leaf morphology and plant architecture in rice

Leaf morphology is an important agronomic trait in rice breeding. We isolated three allelic mutants of NARROW AND ROLLED LEAF 1 (nrl1) which showed phenotypes of reduced leaf width and semi-rolled leaves and different degrees of dwarfism. Microscopic analysis indicated that the nrl1-1 mutant had fewer longitudinal veins and smaller adaxial bulliform cells compared with the wild-type. The NRL1 gene was mapped to the chromosome 12 and encodes the cellulose synthase-like protein D4 (OsCslD4). Sequence analyses revealed single base substitutions in the three allelic mutants. Genetic complementation and over-expression of the OsCslD4 gene confirmed the identity of NRL1. The gene was expressed in all tested organs of rice at the heading stage and expression level was higher in vigorously growing organs, such as roots, sheaths and panicles than in elsewhere. In the mutant leaves, however, the expression level was lower than that in the wild-type. We conclude that OsCslD4 encoded by NRL1 plays a critical role in leaf morphogenesis and vegetative development in rice.     

SV40灭活疫苗的制备及其对小鼠免疫的研究

猿猴空泡病毒40(Simian vacuolating virus 40,SV40) 属于乳多空病毒科,是一种DNA肿瘤病毒。亚洲猿类特别是恒河猴是SV40的天然宿主。感染SV40病毒可导致猴体急性病变或呈长期带毒状态,此外能诱使幼鼠产生肿瘤,并能使多种培养细胞发生转化。本研究初步建立了SV40 病毒在Vero细胞中的增殖培养方法,并且初步建立了β丙内脂灭活病毒的方法和纯化工艺。使用SV40病毒灭活疫苗对Balb/c小鼠进行了免疫,结果表明该疫苗具有较好的免疫原性。随后对SV40 病毒DNA在免疫小鼠的重要脏器中的整合情况进行了调查,结果表明SV40病毒DNA未在小鼠重要脏器中整合。本研究为SV40病毒灭活疫苗的研制和进一步开展猴体抗SV40 感染实验奠定了良好的基础。     

The PDZ-GEF Gef26 regulates synapse development and function via FasII and Rap1 at the Drosophila neuromuscular junction

Guanine nucleotide exchange factors (GEFs) are essential for small G proteins to activate their downstream signaling pathways, which are involved in morphogenesis, cell adhesion, and migration. Mutants of Gef26, a PDZ-GEF (PDZ domain-containing guanine nucleotide exchange factor) in Drosophila, exhibit strong defects in wings, eyes, and the reproductive and nervous systems. However, the precise roles of Gef26 in development remain unclear. In the present study, we analyzed the role of Gef26 in synaptic development and function. We identified significant decreases in bouton number and branch length at larval neuromuscular junctions (NMJs) in Gef26 mutants, and these defects were fully rescued by restoring Gef26 expression, indicating that Gef26 plays an important role in NMJ morphogenesis. In addition to the observed defects in NMJ morphology, electrophysiological analyses revealed functional defects at NMJs, and locomotor deficiency appeared in Gef26 mutant larvae. Furthermore, Gef26 regulated NMJ morphogenesis by regulating the level of synaptic Fasciclin II (FasII), a well-studied cell adhesion molecule that functions in NMJ development and remodeling. Finally, our data demonstrate that Gef26-specific small G protein Rap1 worked downstream of Gef26 to regulate the level of FasII at NMJs, possibly through a βPS integrin-mediated signaling pathway. Taken together, our findings define a novel role of Gef26 in regulating NMJ development and function.     

Numerical simulation of two-phase non-Newtonian blood flow with fluid-structure interaction in aortic dissection

The behavior of blood cells and vessel compliance significantly influence hemodynamic parameters, which are closely related to the development of aortic dissection. Here the two-phase non-Newtonian model and the fluid-structure interaction (FSI) method are coupled to simulate blood flow in a patient-specific dissected aorta. Moreover, three-element Windkessel model is applied to reproduce physiological pressure waves. Important hemodynamic indicators, such as the spatial distribution of red blood cells (RBCs) and vessel wall displacement, which greatly influence the hemodynamic characteristics are analyzed. Results show that the proximal false lumen near the entry tear appears to be a vortex zone with a relatively lower volume fraction of RBCs, a low time-averaged wall shear stress (TAWSS) and a high oscillatory shear index (OSI), providing a suitable physical environment for the formation of atherosclerosis. The highest TAWSS is located in the narrow area of the distal true lumen which might cause further dilation. TAWSS distributions in the FSI model and the rigid wall model show similar trend, while there is a significant difference for the OSI distributions. We suggest that an integrated model is essential to simulate blood flow in a more realistic physiological environment with the ultimate aim of guiding clinical treatment.