Three‐dimensional cell culture microarray for high‐throughput studies of stem cell fate

We have developed a novel three‐dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high‐throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox‐1, whose levels were also measured in situ using a GFP reporter system. In addition, the high‐throughput capacity of the platform was tested using a dual‐slide system that allowed rapid screening of the effects of tretinoin and fibroblast growth factor‐4 (FGF‐4) on the pluripotency of mouse ES cells. This high‐throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses. Biotechnol. Bioeng. 2010; 106: 106–118. © 2010 Wiley Periodicals, Inc.     

Protein Tyrosine Phosphatase PTP1B Is Involved in Hippocampal Synapse Formation and Learning

ER-bound PTP1B is expressed in hippocampal neurons, and accumulates among neurite contacts. PTP1B dephosphorylates ß-catenin in N-cadherin complexes ensuring cell-cell adhesion. Here we show that endogenous PTP1B, as well as expressed GFP-PTP1B, are present in dendritic spines of hippocampal neurons in culture. GFP-PTP1B overexpression does not affect filopodial density or length. In contrast, impairment of PTP1B function or genetic PTP1B-deficiency leads to increased filopodia-like dendritic spines and a reduction in mushroom-like spines, while spine density is unaffected. These morphological alterations are accompanied by a disorganization of pre- and post-synapses, as judged by decreased clustering of synapsin-1 and PSD-95, and suggest a dynamic synaptic phenotype. Notably, levels of ß-catenin-Tyr-654 phosphorylation increased ∼5-fold in the hippocampus of adult PTP1B^−/− (KO) mice compared to wild type (WT) mice and this was accompanied by a reduction in the amount of ß-catenin associated with N-cadherin. To determine whether PTP1B-deficiency alters learning and memory, we generated mice lacking PTP1B in the hippocampus and cortex (PTP1B^fl/fl–Emx1-Cre). PTP1B^fl/fl–Emx1-Cre mice displayed improved performance in the Barnes maze (decreased time to find and enter target hole), utilized a more efficient strategy (cued), and had better recall compared to WT controls. Our results implicate PTP1B in structural plasticity within the hippocampus, likely through modulation of N-cadherin function by ensuring dephosphorylation of ß-catenin on Tyr-654. Disruption of hippocampal PTP1B function or expression leads to elongation of dendritic filopodia and improved learning and memory, demonstrating an exciting novel role for this phosphatase.     

Marburg Virus VP35 Can Both Fully Coat the Backbone and Cap the Ends of dsRNA for Interferon Antagonism

Filoviruses, including Marburg virus (MARV) and Ebola virus (EBOV), cause fatal hemorrhagic fever in humans and non-human primates. All filoviruses encode a unique multi-functional protein termed VP35. The C-terminal double-stranded (ds)RNA-binding domain (RBD) of VP35 has been implicated in interferon antagonism and immune evasion. Crystal structures of the VP35 RBD from two ebolaviruses have previously demonstrated that the viral protein caps the ends of dsRNA. However, it is not yet understood how the expanses of dsRNA backbone, between the ends, are masked from immune surveillance during filovirus infection. Here, we report the crystal structure of MARV VP35 RBD bound to dsRNA. In the crystal structure, molecules of dsRNA stack end-to-end to form a pseudo-continuous oligonucleotide. This oligonucleotide is continuously and completely coated along its sugar-phosphate backbone by the MARV VP35 RBD. Analysis of dsRNA binding by dot-blot and isothermal titration calorimetry reveals that multiple copies of MARV VP35 RBD can indeed bind the dsRNA sugar-phosphate backbone in a cooperative manner in solution. Further, MARV VP35 RBD can also cap the ends of the dsRNA in solution, although this arrangement was not captured in crystals. Together, these studies suggest that MARV VP35 can both coat the backbone and cap the ends, and that for MARV, coating of the dsRNA backbone may be an essential mechanism by which dsRNA is masked from backbone-sensing immune surveillance molecules.     

Heat Stress Impedes Development and Lowers Fecundity of the Brown Planthopper Nilaparvata lugens (St?l)

This study investigated the effects of sub-lethal high temperatures on the development and reproduction of the brown plant hopper Nilaparvata lugens (Stål). When first instar nymphs were exposed at their ULT₅₀ (41.8°C) mean development time to adult was increased in both males and females, from 15.2±0.3 and 18.2±0.3 days respectively in the control to 18.7±0.2 and 19±0.2 days in the treated insects. These differences in development arising from heat stress experienced in the first instar nymph did not persist into the adult stage (adult longevity of 23.5±1.1 and 24.4±1.1 days for treated males and females compared with 25.7±1.0 and 20.6±1.1 days in the control groups), although untreated males lived longer than untreated females. Total mean longevity was increased from 38.8±0.1 to 43.4±1.0 days in treated females, but male longevity was not affected (40.9±0.9 and 42.2±1.1 days respectively). When male and female first instar nymphs were exposed at their ULT₅₀ of 41.8°C and allowed to mate on reaching adult, mean fecundity was reduced from 403.8±13.7 to 128.0±16.6 eggs per female in the treated insects. Following exposure of adult insects at their equivalent ULT₅₀ (42.5°C), the three mating combinations of treated male x treated female, treated male x untreated female, and untreated male x treated female produced 169.3±14.7, 249.6±21.3 and 233.4±17.2 eggs per female respectively, all significantly lower than the control. Exposure of nymphs and adults at their respective ULT₅₀ temperatures also significantly extended the time required for their progeny to complete egg development for all mating combinations compared with control. Overall, sub-lethal heat stress inhibited nymphal development, lowered fecundity and extended egg development time.     

Stress and sex influences on food-seeking behaviors

Objective: Stress increases the drive to consume calorically dense preferred foods suggesting an exogenous factor that may induce caloric overconsumption and weight gain. As females show heightened stress sensitivity and present with increased rates of obesity, we hypothesized that stress‐induced increases in the motivation for preferred foods may be a sex‐specific predisposing factor for weight gain. Methods and Procedures: To investigate this hypothesis, we have developed a buried food paradigm that permits the measurement of sex differences and effects of chronic variable stress (CVS) on the latency to uncover and the consumption of a preferred food pellet without the requisite caloric restriction required in traditional operant conditioning tasks. Results: In our studies, females consistently showed latencies that were twice as fast as males to locate the buried pellet in limited access tests. Interestingly, during stress exposure, male latencies decreased to that of control female levels. Male and female mice showed a significant effect of stress, three‐ and fourfold, respectively, on increased consumption of the preferred food during testing. Discussion: These results support a basal sex difference in behaviors toward a preferred food, and a possible role of stress sensitivity in the drive and intake of such foods. Sex differences in the role stress plays in these behaviors may provide insight into underlying mechanisms related to an increased obesity risk.     

Efficient parallel synthesis of macrocyclic peptidomimetics

A new method for solid phase parallel synthesis of chemically and conformationally diverse macrocyclic peptidomimetics is reported. A key feature of the method is access to broad chemical and conformational diversity. Synthesis and mechanistic studies on the macrocyclization step are reported.     

Pattern of expression of HtrA1 during mouse development.

The human HtrA family of proteases consists of four members: HtrA1, HtrA2, HtrA3, and HtrA4. In humans the four HtrA homologues appear to be involved in several important functions such as cell growth, apoptosis, and inflammatory reactions, and they control cell fate via regulated protein metabolism. In previous studies it was shown that the expression of HtrA1 was ubiquitous in normal adult human tissues. Here we examined the expression of HtrA1 protein and its corresponding mRNA during mouse embryogenesis using Northern blotting hybridization, RT-PCR, and immunohistochemical staining analyses. Our results indicate that HtrA1 is expressed in a variety of tissues in mouse embryos. Furthermore, this expression is regulated in a spatial and temporal manner. Relatively low levels of HtrA1 mRNA are detected in embryos at the beginning of organogenesis (E8), and the levels of expression increase during late organogenesis (E14-E19). Our results show that HtrA1 was expressed during embryonic development in specific areas where signaling by TGFbeta family proteins plays important regulatory roles. The expression of HtrA1, documented both at mRNA and protein levels by RT-PCR and immunohistochemistry in the developing nervous system, is consistent with a possible role of this protein both in dividing and postmitotic neurons, possibly via its documented inhibitory effects on TGFbeta proteins. An exhaustive knowledge of the different cell- and tissue-specific patterns of expression of HtrA1 in normal mouse embryos is essential for a critical evaluation of the exact role played by this protein during development.     

Developmental thresholds and day-degree requirements of Paratanytarsus grimmii and Corynoneura scutellata (Diptera: Chironomidae): two midges associated with potable water treatment

Abstract.  Studies on the development and mortality of Paratanytarsus grimmii (Schneider) and Corynoneura scutellata (Winnertz), which can infest potable water distribution networks, were carried out at eight constant temperatures between 10 °C and 27 °C. Using weighted linear regression, the developmental thresholds were calculated as 7.9 and 8.7 °C for P. grimmii and C. scutellata , respectively. Corynoneura scutellata had a day-degree requirement of 172.4 per generation and developed significantly faster ( P  < 0.001) than P. grimmii with a day-degree requirement of 294.1. Both species were identified as multivoltine with a maximum theoretical number of five generations per year for P. grimmii and seven for C. scutellata . These results are discussed in the context of the management of chironomid infestations within water-treatment works.