Patterns of dye coupling involving serotonergic neurons provide insights into the cellular organization of a central complex lineage of the embryonic grasshopper Schistocerca gregaria

All eight neuroblasts from the pars intercerebralis of one protocerebral hemisphere whose progeny contribute fibers to the central complex in the embryonic brain of the grasshopper Schistocerca gregaria generate serotonergic cells at stereotypic locations in their lineages. The pattern of dye coupling involving these neuroblasts and their progeny was investigated during embryogenesis by injecting fluorescent dye intracellularly into the neuroblast and/or its progeny in brain slices. The tissue was then processed for anti-serotonin immunohistochemistry. A representative lineage, that of neuroblast 1-3, was selected for detailed study. Stereotypic patterns of dye coupling were observed between progeny of the lineage throughout embryogenesis. Dye injected into the soma of a serotonergic cell consistently spread to a cluster of between five and eight neighboring non-serotonergic cells, but never to other serotonergic cells. Dye injected into a non-serotonergic cell from such a cluster spread to other non-serotonergic cells of the cluster, and to the immediate serotonergic cell, but never to further serotonergic cells. Serotonergic cells tested from different locations within the lineage repeat this pattern of dye coupling. All dye coupling was blocked on addition of an established gap junctional blocker (n-heptanol) to the bathing medium. The lack of coupling among serotonergic cells in the lineage suggests that each, along with its associated cluster of dye-coupled non-serotonergic cells, represents an independent communicating pathway (labeled line) to the developing central complex neuropil. The serotonergic cell may function as the coordinating element in such a projection system.     

MicroRNA-9 reveals regional diversity of neural progenitors along the anterior-posterior axis

Neural progenitors self-renew and generate neurons throughout the central nervous system. Here, we uncover an unexpected regional specificity in the properties of neural progenitor cells, revealed by the function of a microRNA--miR-9. miR-9 is expressed in neural progenitors, and its knockdown results in an inhibition of neurogenesis along the anterior-posterior axis. However, the underlying mechanism differs--in the hindbrain, progenitors fail to exit the cell cycle, whereas in the forebrain they undergo apoptosis, counteracting the proliferative effect. Among several targets, we functionally identify hairy1 as a primary target of miR-9, regulated at the mRNA level. hairy1 mediates the effects of miR-9 on proliferation, through Fgf8 signaling in the forebrain and Wnt signaling in the hindbrain, but affects apoptosis only in the forebrain, via the p53 pathway. Our findings show a positional difference in the responsiveness of progenitors to miR-9 depletion, revealing an underlying divergence of their properties.     

Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA

RIG-I is a key innate immune pattern-recognition receptor that triggers interferon expression upon detection of intracellular 5'triphosphate double-stranded RNA (5'ppp-dsRNA) of viral origin. RIG-I comprises N-terminal caspase activation and recruitment domains (CARDs), a DECH helicase, and a C-terminal domain (CTD). We present crystal structures of the ligand-free, autorepressed, and RNA-bound, activated states of RIG-I. Inactive RIG-I has an open conformation with the CARDs sequestered by a helical domain inserted between the two helicase moieties. ATP and dsRNA binding induce a major rearrangement to a closed conformation in which the helicase and CTD bind the blunt end 5'ppp-dsRNA with perfect complementarity but incompatibly with continued CARD binding. We propose that after initial binding of 5'ppp-dsRNA to the flexibly linked CTD, co-operative tight binding of ATP and RNA to the helicase domain liberates the CARDs for downstream signaling. These findings significantly advance our molecular understanding of the activation of innate immune signaling helicases.     

A cellular network of dye-coupled glia associated with the embryonic central complex in the grasshopper Schistocerca gregaria

The central complex of the grasshopper (Schistocerca gregaria) brain comprises a modular set of neuropils, which develops after mid-embryogenesis and is functional on hatching. Early in embryogenesis, Repo-positive glia cells are found intermingled among the commissures of the midbrain, but then redistribute as central complex modules become established and, by the end of embryogenesis, envelop all midbrain neuropils. The predominant glia associated with the central body during embryogenesis are glutamine synthetase-/Repo-positive astrocyte-like glia, which direct extensive processes (gliopodia) into and around midbrain neuropils. We used intracellular dye injection in brain slices to ascertain whether such glia are dye-coupled into a communicating cellular network during embryogenesis. Intracellular staining of individual cells located at any one of four sites around the central body revealed a population of dye-coupled cells whose number and spatial distribution were stereotypic for each site and comparable at both 70 and 100% of embryogenesis. Subsequent immunolabeling confirmed these dye-coupled cells to be astrocyte-like glia. The addition of n-heptanol to the bathing saline prevented all dye coupling, consistent with gap junctions linking the glia surrounding the central body. Since dye coupling also occurred in the absence of direct intersomal contacts, it might additionally involve the extensive array of gliopodia, which develop after glia are arrayed around the central body. Collating the data from all injection sites suggests that the developing central body is surrounded by a network of dye-coupled glia, which we speculate may function as a positioning system for the developing neuropils of the central complex.     

基于CERES-Rice模型的湖南省一季稻极端高温损失评估及适应性措施

以湖南省4个农业气象站点的一季稻为研究对象,基于1990-2012年逐日气象数据,精细的土壤以及田间管理记录,分析了过去23年极端高温的变化趋势,并利用校准后的CERES-Rice模型评价了高温热害的致损率,着重探讨了不同的适应性措施对缓解高温热害的作用,以期提出科学合理的减灾措施来保障我国的粮食安全。结果表明：（1）CERES-Rice能很好地捕捉本研究区不同天气和管理条件下的水稻物候期和产量,除桑植站开花期外,其余各参数的模拟误差均小于10%。（2）一季稻生育期内极端高温频发且在本世纪有增强趋势,灾损率由高到低依次为古丈 > 桑植 > 怀化 > 靖州,分别为10.4%,8.2%,7.5%和4%。随着气候变暖的加剧,一季稻生产将面临着日趋严重的高温热害风险。（3）选用耐高温品种产量最大可提升29.8%,但在极端高温年份,提高品种高温抗性的方法收效甚微。调节播种期会导致-25%-20%的产量波动,其中提前10 d或5 d种植均可缓解极端高温的危害。增加灌溉的贡献为1%-8%,其中6-8 cm灌水量效果最佳。极端高温期间增施氮肥的贡献稳定且显著,平均增产2%-20%,80-100 kg/hm²的施氮量能带来较为理想的避热增产效果。     

The conformational state of Tes regulates its zyxin-dependent recruitment to focal adhesions

The function of the human Tes protein, which has extensive similarity to zyxin in both sequence and domain organization, is currently unknown. We now show that Tes is a component of focal adhesions that, when expressed, negatively regulates proliferation of T47D breast carcinoma cells. Coimmunoprecipitations demonstrate that in vivo Tes is complexed with actin, Mena, and vasodilator-stimulated phosphoprotein (VASP). Interestingly, the isolated NH2-terminal half of Tes pulls out alpha-actinin and paxillin from cell extracts in addition to actin. The COOH-terminal half recruits zyxin as well as Mena and VASP from cell extracts. These differences suggest that the ability of Tes to associate with alpha-actinin, paxillin, and zyxin is dependent on the conformational state of the molecule. Consistent with this hypothesis, we demonstrate that the two halves of Tes interact with each other in vitro and in vivo. Using fibroblasts lacking Mena and VASP, we show that these proteins are not required to recruit Tes to focal adhesions. However, using RNAi ablation, we demonstrate that zyxin is required to recruit Tes, as well as Mena and VASP, but not vinculin or paxillin, to focal adhesions.     

Inorganic phosphate modulates responsiveness to 24,25(OH)2D3 in chondrogenic ATDC5 cells

Chondrogenic ATDC5 cells were used as a model of in vitro endochondral maturation to study the role of inorganic phosphate (Pi) in the regulation of growth plate chondrocytes by vitamin D3 metabolites. ATDC5 cells that were cultured for 10 days post‐confluence in differentiation media and then treated for 24 h with Pi produced a type II collagen matrix based on immunohistochemistry and expressed mRNAs for several chondrocytic markers, including aggrecan, collagen types II and X, cartilage oligomeric matrix protein, and SOX9. Pi also caused a decrease in [³⁵S]‐sulfate incorporation and stimulated apoptosis, as evidenced by increased DNA fragmentation and caspase‐3 activity. In addition, treatment with Pi induced sensitivity to 24,25‐dihydroxyvitamin D3 and this effect was both dose‐dependent and was blocked by phosphonoformic acid (PFA), a specific inhibitor of sodium dependent type III Pi transporters. Treatment with 24R,25(OH)₂D₃ reduced cell number and increased alkaline phosphatase specific activity in a dose‐dependent manner. Moreover, 24R,25(OH)₂D₃ reversed the Pi‐induced decrease in incorporation of [³H]‐thymidine and [³⁵S]‐sulfate incorporation, as well as the Pi‐induced increase in apoptosis. These results suggest that Pi acts as an early chondrogenic differentiation factor, inducing response to 24R,25(OH)₂D₃; treatment of committed chondrocytes with Pi induces apoptosis, but 24R,25(OH)₂D₃ mitigates these effects, indicating a possible inhibitory feedback loop. J. Cell. Biochem. 107: 155–162, 2009. © 2009 Wiley‐Liss, Inc.     

Controlling glycation of recombinant antibody in fed-batch cell cultures

Protein glycation is a non-enzymatic glycosylation that can occur to proteins in the human body, and it is implicated in the pathogenesis of multiple chronic diseases. Glycation can also occur to recombinant antibodies during cell culture, which generates structural heterogeneity in the product. In a previous study, we discovered unusually high levels of glycation (>50%) in a recombinant monoclonal antibody (rhuMAb) produced by CHO cells. Prior to that discovery, we had not encountered such high levels of glycation in other in-house therapeutic antibodies. Our goal here is to develop cell culture strategies to decrease rhuMAb glycation in a reliable, reproducible, and scalable manner. Because glycation is a post-translational chemical reaction between a reducing sugar and a protein amine group, we hypothesized that lowering the concentration of glucose--the only source of reducing sugar in our fed-batch cultures--would lower the extent of rhuMAb glycation. When we decreased the supply of glucose to bioreactors from bolus nutrient and glucose feeds, rhuMAb glycation decreased to below 20% at both 2-L and 400-L scales. When we maintained glucose concentrations at lower levels in bioreactors with continuous feeds, we could further decrease rhuMAb glycation levels to below 10%. These results show that we can control glycation of secreted proteins by controlling the glucose concentration in the cell culture. In addition, our data suggest that rhuMAb glycation occurring during the cell culture process may be approximated as a second-order chemical reaction that is first order with respect to both glucose and non-glycated rhuMAb. The basic principles of this glycation model should apply to other recombinant proteins secreted during cell culture.