Investigating design principles of micropatterned encapsulation systems containing high-density microtissue arrays

Immunoisolation is an important strategy to protect transplanted cells from rejection by the host immune system.Recently,microfabrication techniques have been used to create hydrogel membranes to encapsulate microtissue in an arrayed organization.The method illustrates a new macroencapsulation paradigm that may allow transplantation of a large number of cells with microscale spatial control,while maintaining an encapsulation device that is easily maneuverable and remaining integrated following transplantation.This study aims to investigate the design principles that relate to the translational application of micropatterned encapsulation membranes,namely,the control over the transplantation density/quantity of arrayed microtissues and the fidelity of pre-formed microtissues to micropatterns.Agarose hydrogel membranes with microwell patterns were used as a model encapsulation system to exemplify these principles.Our results show that high-density micropatterns can be generated in hydrogel membranes,which can potentially maximize the percentage volume of cellular content and thereby the transplantation efficiency of the encapsulation device.Direct seeding of microtissues demonstrates that microwell structures can efficiently position and organize pre-formed microtissues,suggesting the capability of micropatterned devices for manipulation of cellular transplants at multicellular or tissue levels.Detailed theoretical analysis was performed to provide insights into the relationship between micropatterns and the transplantation capacity of membrane-based encapsulation.Our study lays the ground for developing new macroencapsulation systems with microscale cellular/tissue patterns for regenerative transplantation.     

Role of Tim50 in the Transfer of Precursor Proteins from the Outer to the Inner Membrane of Mitochondria

Transport of essentially all matrix and a number of inner membrane proteins is governed, entirely or in part, by N-terminal presequences and requires a coordinated action of the translocases of outer and inner mitochondrial membranes (TOM and TIM23 complexes). Here, we have analyzed Tim50, a subunit of the TIM23 complex that is implicated in transfer of precursors from TOM to TIM23. Tim50 is recruited to the TIM23 complex via Tim23 in an interaction that is essentially independent of the rest of the translocase. We find Tim50 in close proximity to the intermembrane space side of the TOM complex where it recognizes both types of TIM23 substrates, those that are to be transported into the matrix and those destined to the inner membrane, suggesting that Tim50 recognizes presequences. This function of Tim50 depends on its association with TIM23. We conclude that the efficient transfer of precursors between TOM and TIM23 complexes requires the concerted action of Tim50 with Tim23.     

小麦和大豆茬口对黄瓜土壤微生物生态特征的影响

采用常规稀释平板法和BIOLOG ECO微平板反应系统,研究了小麦和大豆茬口对黄瓜土壤微生物生态特征的影响.结果表明,两种茬口均显著提高了黄瓜土壤微生物真菌、细菌和放线菌的数量,显著降低了尖孢镰刀菌黄瓜专化型的数量(P<0.05);显著提高了土壤微生物群落的Shannon-Wiener指数、均匀度指数、Simpson指数和McIntosh指数(P<0.05)、以及土壤微生物生物量碳,降低了土壤基础呼吸和代谢商(P<0.05),改变了土壤微生物对单一碳源的利用能力.此外,两种茬口还显著提高了土壤中速效磷和速效钾含量,以及黄瓜产量.说明小麦和大豆茬口改善了土壤微生态环境.     

Selection of vimentin-specific antibodies from the HuCAL phage display library by subtractive panning on formalin-fixed, paraffin-embedded tissue

We describe the direct isolation of specific antibodies on formalin-fixed, paraffin-embedded (FFPE) tissue. The technique involves subtractive selection of a large and highly diverse combinatorial human antibody phage library (HuCAL) on lymphocyte FFPE tissue sections. Tissue sections from normal human tonsil tissue were used to deplete the library of binders to most housekeeping proteins. Mantle-cell lymphoma tissue was used for positive selection and enrichment of mantle cell or tumor-specific antibody phage. We established a high-throughput immunohistochemical method for screening of antibody clones selected from FFPE tissue. One recombinant antibody showed specific staining for interfollicular and mantle cells in FFPE tissue. Immunoprecipitation with this antibody and subsequent mass spectrometric analysis revealed specificity for vimentin.     

Disruption of Smad4 in neural crest cells leads to mid-gestation death with pharyngeal arch, craniofacial and cardiac defects

TGFβ/BMP signaling pathways are essential for normal development of neural crest cells (NCCs). Smad4 encodes the only common Smad protein in mammals, which is a critical nuclear mediator of TGFβ/BMP signaling. In this work, we sought to investigate the roles of Smad4 for development of NCCs. To overcome the early embryonic lethality of Smad4 null mice, we specifically disrupted Smad4 in NCCs using a Cre/loxP system. The mutant mice died at mid-gestation with defects in facial primordia, pharyngeal arches, outflow tract and cardiac ventricles. Further examination revealed that mutant embryos displayed severe molecular defects starting from E9.5. Expression of multiple genes, including Msx1, 2, Ap-2α, Pax3, and Sox9, which play critical roles for NCC development, was downregulated by NCC disruption of Smad4. Moreover, increased cell death was observed in pharyngeal arches from E10.5. However, the cell proliferation rate in these areas was not substantially altered. Taken together, these findings provide compelling genetic evidence that Smad4-mediated activities of TGFβ/BMP signals are essential for appropriate NCC development.