Species delineation using Bayesian model-based assignment tests: a case study using Chinese toad-headed agamas (genus <Emphasis Type="Italic">Phrynocephalus</Emphasis>)

Background  

Species are fundamental units in biology, yet much debate exists surrounding how we should delineate species in nature. Species discovery now requires the use of separate, corroborating datasets to quantify independently evolving lineages and test species criteria. However, the complexity of the speciation process has ushered in a need to infuse studies with new tools capable of aiding in species delineation. We suggest that model-based assignment tests are one such tool. This method circumvents constraints with traditional population genetic analyses and provides a novel means of describing cryptic and complex diversity in natural systems. Using toad-headed agamas of the Phrynocephalus vlangalii complex as a case study, we apply model-based assignment tests to microsatellite DNA data to test whether P. putjatia, a controversial species that closely resembles P. vlangalii morphologically, represents a valid species. Mitochondrial DNA and geographic data are also included to corroborate the assignment test results.     

Feasibility of culturing C2C12 mouse myoblasts on glass microcarriers in a continuous stirred tank bioreactor

Commercial culturing of mammalian cell lines is increasing in importance as more biological products unique to mammals are being produced in genetically altered mammalian cells. Most mammalian cells are anchorage dependent, so they must be cultured on a support matrix. This limitation, along with the requirement of a low shear environment, severely effects the scale-up of bench-scale culture systems. The need to culture mammalian cells on a support matrix limits the increase in cell population to a factor of 10-20 before growth virtually stops due to contact inhibition. Commercial culturing systems for anchorage dependent cells are batch processes because of the combination of contact inhibition and support matrix requirements. Development of a continuous bioreactor system could allow both unlimited scale-up and continuous cell-mass production. To design a continuous reactor, a mathematical model to predict the reactor performance should be developed. This paper addresses the development of a mathematical model for predicting continuous bioreactor performance. It was found that anchorage dependent C2C12 mouse myoblast cells, a continuous cell line, followed Monod kinetics for glucose consumption and cell mass production in batch flask experiments, with w_max = 0.040 hr^у and K_m = 2.5 mM. Furthermore, it was found that these parameters could be used to predict the glucose consumption in a continuous bioreactor operated with constant feed of seeded microcarriers operated at two different residence times. The success of this model implies the possibility of developing a continuous cell harvesting and reinoculation system using a microcarrier bioreactor to produce cell mass.     

Use of Genome Engineering to Create Patient Specific MLL Translocations in Primary Human Hematopoietic Stem and Progenitor Cells

One of the challenging questions in cancer biology is how a normal cell transforms into a cancer cell. There is strong evidence that specific chromosomal translocations are a key element in this transformation process. Our studies focus on understanding the developmental mechanism by which a normal stem or progenitor cell transforms into leukemia. Here we used engineered nucleases to induce simultaneous specific double strand breaks in the MLL gene and two different known translocation partners (AF4 and AF9), which resulted in specific chromosomal translocations in K562 cells as well as primary hematopoietic stem and progenitor cells (HSPCs). The initiation of a specific MLL translocation in a small number of HSPCs likely mimics the leukemia-initiating event that occurs in patients. In our studies, the creation of specific MLL translocations in CD34+ cells was not sufficient to transform cells in vitro. Rather, a variety of fates was observed for translocation positive cells including cell loss over time, a transient proliferative advantage followed by loss of the clone, or a persistent proliferative advantage. These studies highlight the application of genome engineering tools in primary human HSPCs to induce and prospectively study the consequences of initiating translocation events in leukemia pathogenesis.     

益生菌混合发酵条件及其对发酵胡萝卜汁色泽及风味的影响

目的 研究3株益生菌混合发酵胡萝卜汁的发酵条件对色泽、风味的影响及其贮藏特性等.方法 通过菌种配比、单因素试验及正交试验优化了胡萝卜汁的发酵条件,并利用分光测色计和气质联用仪分别研究了发酵前后胡萝卜汁的风味成分和色泽变化.结果 添加30％ (m/m)的新鲜胡萝卜汁(原液),接种3％的以1∶1∶1(v/v/v)混合的嗜热...     

[125I]RTI-55 Binding to Cocaine-Sensitive Dopaminergic and Serotonergic Uptake Sites in the Human Brain

[¹²⁵I]RTI-55 is a newly synthesized cocaine congener that may offer advantages over other ligands previously used to examine cocaine binding sites. However, the in vitro pharmacological and anatomical characterization of [¹²⁵I]RTI-55 binding sites has not been previously performed in human brain. To determine the specificity, stability, and feasibility of [¹²⁵I]RTI-55 for use in radioligand binding assays in postmortem human tissue, a series of experiments were performed characterizing [¹²⁵I]RTI-55 binding sites in human brain using homogenized membrane preparations and quantitative autoradtography. Analysis of the association, dissociation, and saturation data favored two-phase processes. A curve-fitting analysis of the data derived in saturation experiments found a high-affinity site with K_D= 66 ± 35 pM and S_max= 13.2 ± 10.1 pmol/g of tissue and a low-affinity site with K_D= 1.52 ± 0.55 nM and B_max of 47.5 ± 11-2 pmol/g of tissue. Competition by ligands known to bind to the dopamine transporter showed a rank order of RTI-55 > GBR-12909 > mazindol > WIN 35428 > = methylphenidate > (?)-cocaine > buproprion > (±)-amphetamine. Binding to serotonergic sites was evaluated in the midbrain. Results of the saturation experiment performed autoradiographically in the midbrain showed a single site with K_D= 370 ± 84 pM. It appears that [¹²⁵I]RTI-55 should be useful in further studies of the regulation of cocaine binding sites using postmortem human specimens.     

The effective population size of Anopheles gambiae in Kenya: implications for population structure

We estimated current and long-term effective population size (Ne) of two Anopheles gambiae (savanna cytotype) populations in Kenya. Temporal variation at nine microsatellite loci in each population sampled 7 and 9 years apart and genetic diversity in each sample were analyzed to answer the following questions. (1) Do bottlenecks occur in Kenyan populations of A. gambiae? (2) How variable are different populations with respect to their current and long-term Ne values? (3) What are the implications of these results on population structure and history? The estimates of Ne of Asembo and Jego were 6,359 and 4,258, respectively, and the lower 95% limits were 2,455 and 1,669, respectively. Thus, despite the typical observation of low density at the village level during the dry season, large populations are maintained annually. Large current Ne is consistent with previous studies showing low differentiation across the continent, especially under Wright's isolation-by-distance model. Current Ne in Asembo was 1.5-fold higher than in Jego, but this difference was not significant. Long-term Ne in Asembo (22,667) was 2.9-fold higher than that in Jego (7,855) based on the stepwise mutation model. The difference between populations was significant at both time points regardless of whether long-term Ne values were calculated based on the stepwise mutation model or the infinite-alleles model. Heterozygosity in Jego declined significantly between 1987 (59%) and 1996 (54%), whereas heterozygosity in Asembo was stable (66%-65%). Despite the relatively high and significant differentiation between Asembo and Jego (FST = 0.072-0.10, RST = 0.037- 0.038), all alleles in Jego were found in Asembo but not vice versa. All of these findings suggest that lower Ne in Jego magnifies differentiation between the two populations. The long-term Ne was biased downward, because its calculation was based on an upper bound estimate of microsatellite mutation rate. Ne values based on mtDNA and allozymes were an order of magnitude higher. Long-term Ne therefore, is probably measured in hundreds of thousands and hence does not support a recent expansion of this species from a small population.      

Peripheral hyaline blebs (podosomes) of macrophages

The plasmalemma and hyaline ectoplasm together constitute the sensory and motor organ of macrophages. The purpose of this study was to isolate this cell fraction in order to analyze it biochemically and functionally. Brief sonification of warmed rabbit lung macrophages caused release of heterodisperse hyaline blebs and filopodia, which were easily collected by differential centrifugation. Viewed in the electron microscope, these structures consisted of membrane-bounded sacs principally containing actin filaments. Some contained secondary lysosomes. They were enriched threefold over whole cell homogenates in specific adenylate cyclase activity and in trichloroacetic-acid-precipitable (125)I when derived from cells labeled with 125(I) by means of a lactoperoxidase-catalyzed reaction. These markers were found to have identical isopycnic densitites when macrophage homogenates were subjected to sedimentation in a focusing sucrose density gradient system, and these markers had densities distinct from those of other cytoplasmic organelles. These markers were therefore assumed to be associated with macrophage plasma membranes. The specific β- glucuronidase activity of the bleb fraction was similar to that of homogenates, but the blebs had considerably lower specific succinic dehydrogenase activity and RNA content, and DNA was undetectable. Electrophoresis of blebs solubilized in sodium dodecyl sulfate on polyacrylamide gels revealed polypeptides co-migrating with macrophage actin-binding protein, myosin, and actin; blebs also had EDTA-activated adenosine triphosphatase activity characteristic of myosin. The concentrations of actin-binding protein and myosin were higher in blebs than in cells or cytoplasmic extracts, whereas actin concentrations were similar (relative to extracts) or only slightly greater (than in cells). Blebs and intact cells had high lactate dehydrogenase activities in the presence but not the absence of Triton X-100. Blebs and cells oxidased 1-[(14)C]glucose, and the rate of glucose oxidation was increased substantially in the presence of latex beads. We conclude that intact sacs of plasmalemma encasing contractile proteins and cytoplasmic enzymes can be isolated from macrophages. They are enriched in myosin and actin-binding protein, indicating that the contractile apparatus is regulated in the cell periphery. These structures have the capacity to respond to environmental signals. We suggest the name "podosomes" for them because of their resemblance to macrophage pseudopodia. We propose that podosome formation results from rapid dissolution of the cortical gel when the membrane is in an actively extended configuration.