GSK-3 signaling in developing cortical neurons is essential for radial migration and dendritic orientation

GSK-3 is an essential mediator of several signaling pathways that regulate cortical development. We therefore created conditional mouse mutants lacking both GSK-3α and GSK-3β in newly born cortical excitatory neurons. Gsk3-deleted neurons expressing upper layer markers exhibited striking migration failure in all areas of the cortex. Radial migration in hippocampus was similarly affected. In contrast, tangential migration was not grossly impaired after Gsk3 deletion in interneuron precursors. Gsk3-deleted neurons extended axons and developed dendritic arbors. However, the apical dendrite was frequently branched while basal dendrites exhibited abnormal orientation. GSK-3 regulation of migration in neurons was independent of Wnt/β-catenin signaling. Importantly, phosphorylation of the migration mediator, DCX, at ser327, and phosphorylation of the semaphorin signaling mediator, CRMP-2, at Thr514 were markedly decreased. Our data demonstrate that GSK-3 signaling is essential for radial migration and dendritic orientation and suggest that GSK-3 mediates these effects by phosphorylating key microtubule regulatory proteins.DOI: http://dx.doi.org/10.7554/eLife.02663.001eLife digestIn the brain, one of the most striking features of the cerebral cortex is that its neurons are organized into different layers that are specifically connected to one another and to other regions of the brain. How newly generated neurons find their appropriate layer during the development of the brain is an important question; and, in humans, when this process goes awry, it can often result in seizures and mental retardation.An enzyme called GSK-3 regulates several major signaling pathways important to brain development. The GSK-3 enzyme switches other proteins on or off by adding phosphate groups to them.Morgan-Smith et al. set out to better understand the role of GSK-3 in brain development by deleting the genes for this enzyme specifically in the cerebral cortex of mice. Mice have two genes that encode slightly different forms of the GSK-3 enzyme. Deleting both of these in different groups of neurons during brain development revealed that a major group of neurons need GSK-3 in order to migrate to the correct layer. Specifically, the movement of neurons from where they arise in the central region of the brain to the outermost layer (a process called radial migration) was disrupted when the GSK-3 genes were deleted.Morgan-Smith et al. further found that cortical neurons without GSK-3 were unable to develop the shape needed to undertake radial migration because they failed to switch from having many branches to having just two main branches. Additional experiments revealed that these abnormalities did not depend on certain signaling pathways, such as the Wnt-signaling pathway or the PI3K signaling pathway that can control GSK-3 activity.Instead, Morgan-Smith et al. found that two proteins that are normally targeted by the GSK-3 enzyme have fewer phosphate groups than normal in the cortical neurons that did not contain the enzyme: both of these proteins regulate the shape of neurons by interacting with the molecular ‘scaffolding’ within the cell. The GSK-3 enzyme was already known to modify the activities of many other proteins that affect the migration of cells. Thus, the findings of Morgan-Smith et al. suggest that this enzyme may coordinate many of the mechanisms thought to underlie this process during brain development.DOI: http://dx.doi.org/10.7554/eLife.02663.00

Probiotic Lactobacillus rhamnosus GG Induces Alterations in Ileal Microbiota With Associated CD3-CD19-T-bet+IFNγ+/- Cell Subset Homeostasis in Pigs Challenged With Salmonella enterica Serovar 4,[5],12:i:-

Salmonella enterica serovar 4,[5],12:i:- (S. 4,[5],12:i:-) is an emerging foodborne pathogen causing salmonellosis in humans and animals. Probiotic Lactobacillus rhamnosus GG (LGG) is an effective strategy for controlling enteric infections through maintaining gut microbiota homeostasis and regulating the intestinal innate immune response. Here, LGG was orally administrated to newly weaned piglets for 1 week before S. 4,[5],12:i:- challenge. S. 4,[5],12:i:- challenge led to disturbed gut microbiota, characterized by increased levels of Psychrobacter, Chryseobacterium indoltheticum, and uncultured Corynebacteriaceae populations, as well as an aberrant correlation network in Prevotellaceae NK3B31 group-centric species. The beneficial effect of LGG correlated with attenuating the expansion of Prevotellaceae NK3B31 group. Fusobacterium only found in the pigs treated with LGG was positively correlated with Lactobacillus animalis and Propionibacterium. Administration of LGG induced the expansion of CD3-CD19-T-bet+IFNγ+ and CD3-CD19-T-bet+IFNγ- cell subsets in the peripheral blood at 24 h after a challenge of S. 4,[5],12:i:-. S. 4,[5],12:i:- infection increased the population of intraepithelial CD3-CD19-T-bet+IFNγ+ and CD3-CD19-T-bet+IFNγ- cells in the ileum; however, this increase was attenuated via LGG administration. Correlation analysis revealed that LGG enriched Flavobacterium frigidarium and Facklamia populations, which were negatively correlated with intraepithelial CD3-CD19-T-bet+IFNγ+ and CD3-CD19-T-bet+IFNγ- cells in the ileum. The present data suggest that probiotic LGG alters gut microbiota with associated CD3-CD19-T-bet+IFNγ+/- cell subset homeostasis in pigs challenged with S. enterica 4,[5],12:i:-. LGG may be used in potential gut microbiota-targeted therapy regimens to regulate the specific immune cell function and, consequently, control enteric infections

Synthesis of a Dual Functional Anti-MDR Tumor Agent PH II-7 with Elucidations of Anti-Tumor Effects and Mechanisms

Multidrug resistance mediated by P-glycoprotein in cancer cells has been a major issue that cripples the efficacy of chemotherapy agents. Aimed for improved efficacy against resistant cancer cells, we designed and synthesized 25 oxindole derivatives based on indirubin by structure-activity relationship analysis. The most potent one was named PH II-7, which was effective against 18 cancer cell lines and 5 resistant cell lines in MTT assay. It also significantly inhibited the resistant xenograft tumor growth in mouse model. In cell cycle assay and apoptosis assay conducted with flow cytometry, PH II-7 induced S phase cell cycle arrest and apoptosis even in resistant cells. Consistently revealed by real-time PCR, it modulates the expression of genes related to the cell cycle and apoptosis in these cells, which may contributes to its efficacy against them. By side-chain modification and FITC-labeling of PH II-7, we were able to show with confocal microscopy that not only it was not pumped by P-glycoprotein, it also attenuated the efflux of Adriamycin by P-glycoprotein in MDR tumor cells. Real-time PCR and western blot analysis showed that PH II-7 down-regulated MDR1 gene via protein kinase C alpha (PKCA) pathway, with c-FOS and c-JUN as possible mediators. Taken together, PH II-7 is a dual-functional compound that features both the cytotoxicity against cancer cells and the inhibitory effect on P-gp mediated drug efflux

Early inflammatory response in periparturient sows to experimentally induced Escherichia coli mastitis

The overall objective of the present work was to monitor the induction of some key factors involved in the early inflammatory response in the mammary gland of periparturient sows to experimentally induced Escherichia coli (E. coli) mastitis. We wanted to gain a better understanding of why some sows develop clinical signs of mastitis, while others remain clinically healthy. Concentrations of the proinflammatory cytokines interleukin (IL)-6 and tumour necrosis factor-alpha (TNF-alpha), and the acute-phase protein (APP) serum amyloid A (SAA) in blood increased in sows following intramammary E. coli inoculation as measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, concentrations of IL-6 and TNF-alpha in blood were higher in sows that developed clinical signs of mastitis than in sows that remained clinically healthy after inoculation. Notably, immunohistochemistry (IHC) analysis of biopsy specimen revealed that some baseline production of cytokines took place in normal mammary glands of pregnant sows, and that sows that developed clinical signs of mastitis had significantly lower baseline production of IL-1beta than did sows that remained clinically healthy. Twenty-four hours after inoculation, there was an increase in the production of IL-1beta, IL-6, IL-8 and TNF-alpha in the inoculated mammary glands of sows that developed clinical signs of mastitis. By contrast, sows that remained clinically healthy did not show increased production of IL-1beta, IL-6 and TNF-alpha in the inoculated mammary glands. However, at the mRNA level there was an increase in the expression of IL-1beta and TNF-alpha also in these sows. The anti-inflammatory cytokine IL-10 mRNA expression increased in the inoculated mammary glands 24 hours after inoculation, and it was higher in the inoculated mammary glands of sows that developed clinical signs of mastitis compared with sows that remained clinically healthy, while the expression of another anti-inflammatory cytokine, transforming growth factor-beta 1 (TGF-beta1) mRNA was unaltered, as was mRNA expression for the IL-1 receptor type I (IL-1R1). In addition, the pattern-recognition receptor (PRR) Toll-like receptor 2 (TLR2) mRNA expression increased in the inoculated as well as the non-inoculated mammary glands of sows that developed clinical signs of mastitis and of sows that remained clinically healthy. However, TLR2 mRNA expression was higher in the inoculated mammary glands than in the non-inoculated mammary glands of sows that remained clinically healthy. The findings of the present study suggest that IL-6 and/or TNF-alpha in blood could be used as markers for identification of periparturient sows with coliform mastitis, and that development of clinical symptoms of coliform mastitis is associated with the degree of local production of regulatory cytokines in response to intramammary E. coli inoculation

Research Studies on the Thrust of Special-Shaped Full-Sectional Cutterheads of Quasirectangular Shield

Thrust of shield cutters is the major parameter of tunnel construction and an important index for shield machine design. The thrust bearing resistance of a shield machine has a significant impact on its construction efficiency and safe operation. The use of quasirectangular shield not only can increase the space utilization rate but also avoid the deformation of the back soil when compared with a conventional circular or rectangular shield. In this paper, structural analysis of quasirectangular shield cutterhead is carried out and a corresponding mathematical thrust model is developed. Both the stress and displacement distributions of cutterhead are calculated. It is found that the stress value in most regions of the cutterhead is between 5 MPa and 45 MPa. The maximum stress is 208.44 MPa, which is at the middle part of the rib and is below the yield limit. The maximum deformation is found in the center area of the chest plate, the value of which is also within the design requirement. In addition, a monitoring method suitable for quasirectangular shield is proposed. The appropriateness and reliability of the proposed monitoring method are demonstrated by the comparison between the numerical simulation and monitoring method

Analysis of Tangential Leakage Flow Characteristics of Oil-Free Scroll Expander for a Micro-Scale Compressed Air Energy Storage System

Tangential leakage loss is the primary factor that significantly affects the output performance of oil-free scroll expanders. A scroll expander can function under different operating conditions, and the flow of tangential leakage and generation mechanism is different. This study employed computational fluid dynamics to investigate the unsteady flow characteristics of the tangential leakage flow of a scroll expander with air as the working fluid. Consequently, the effects of different radial gap sizes, rotational speeds, inlet pressures, and temperatures on the tangential leakage were discussed. The tangential leakage decreased with increases in the scroll expander rotational speed, inlet pressure, and temperature, and decreased with decrease in radial clearance. With an equal-proportional increase in radial clearance, the flow form of the gas in the first expansion and back-pressure chambers became more complicated; when the radial clearance increased from 0.2 to 0.5 mm, the volumetric efficiency of the scroll expander decreased by approximately 5.0521%. Moreover, because of the large radial clearance, the tangential leakage flow maintained a subsonic flow. Further, the tangential leakage decreased with increase in rotational speed, and when the rotational speed increased from 2000 to 5000 r/min, the volumetric efficiency increased by approximately 8.7565%

Selection of the Key Segment Position for Trapezoidal Tapered Rings and Calculation of the Range of Jack Stroke Differences with a Predetermined Key Segment Position

It is generally accepted that selecting the key segment position for trapezoidal tapered rings and controlling the shield machine advancement are challenging tasks for shield tunneling projects. In this work, we propose a method for calculating the key segment position based on the shield tail gap, jack stroke difference, and lining trend. To calculate all possible key segment positions other than that corresponding to the straight joint configuration, the shield tail gap that remains after segment assembly and the jack stroke difference corresponding to the advancement of the segmental lining and lining trend were computed; then, values and importance coefficients were assigned to these factors according to current operating conditions. To ensure that the segmental lining can be assembled successfully with the calculated key position, we established a model to calculate the change in the shield tail gap before and after shield machine advancement based on the spatial relationships of the shield machine, the currently installed segmental rings, and the segment to be installed. Further, we propose a method for calculating the range of jack stroke differences when the predetermined “permitted shield tail gap” and key position are provided. The method is based on the change in the shield tail gap calculated with the above model and the positional relationship between the shield machine’s actual axis and the designed tunnel axis after the current segmental ring has been assembled. The calculated range of jack stroke differences may then be used to control the advancement of the shield machine. We validated the viability of our methods by using the data of Phase 1 works on Line 2 of the Ningbo Rail Transit system

Morphometric analysis of proinflammatory cytokines in mammary glands of sows suggests an association between clinical mastitis and local production of IL-1beta, IL-6 and TNF-alpha

Twelve healthy primiparous sows received intramammary inoculation with Escherichia coli (serotype O127) during the 24-h period preceding parturition. Mammary gland biopsy samples were taken immediately before inoculation (0 h) and from the inoculated and the contralateral non-inoculated glands 24 h after inoculation. The analyses of interleukin-1 beta (IL-1$\beta$), IL-6, IL-8, and tumor necrosis factor alpha (TNF-$\alpha$) by immunohistochemistry revealed that the production of these proinflammatory cytokines significantly increased in the inoculated mammary glands of sows that developed clinical signs of mastitis (affected group, $n=4$) 24 h after inoculation. This was also true for IL-8 in the inoculated mammary glands of sows that did not develop clinical signs of mastitis (nonaffected group, $n=8$). Sows that developed clinical signs of mastitis displayed significantly lower constitutive production of IL-1$\beta$ than did sows that remained clinically healthy. The data indicate that the development of clinical symptoms of coliform mastitis in the sow is associated with a locally increased proinflammatory cytokine production in response to intramammary E. coli infection