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.     

Advances in preimplantation genetic diagnosis/screening

Preimplantation genetic diagnosis (PGD) gives couples who have a high risk of transmitting genetic disorders to their baby the chance to have a healthy offspring through embryo genetic analysis and selection. Preimplantation genetic screening (PGS) is an effective method to select euploid embryos that may prevent repeated implantation failure or miscarriage. However, how and to whom PGS should be provided is a controversial topic. The first successful case of PGD of a human being was reported in 1990, and there have been tremendous improvements in this technology since then. Both embryo biopsy and genetic technologies have been improved dramatically, which increase the accuracy and expand the indications of PGD/PGS.     

Parameters and Efficiency of Direct Gene Disruption by Zinc Finger Nucleases in Medaka Embryos

Zinc finger nucleases (ZFNs) can generate targeted gene disruption (GD) directly in developing embryos of zebrafish, mouse and human. In the fish medaka, ZFNs have been attempted on a transgene. Here, we developed procedures and parameters for ZFN-mediated direct GD on the gonad-specifically expressed gsdf locus in medaka. A pair of ZFNs was designed to target the first exon of gsdf and their synthetic mRNAs were microinjected into 1-cell stage embryos. We reveal dose-dependent survival rate and GD efficiency. In fry, ZFN mRNA injection at 10 ng/μl led to a GD efficiency of 30 %. This value increased up to nearly 100 % when the dose was enhanced to 40 ng/μl. In a typical series of experiments of ZFN mRNA injection at 10 ng/μl, 420 injected embryos developed into 94 adults, 4 of which had altered gsdf alleles. This leads to a GD efficacy of ～4 % in the adulthood. Sequencing revealed a wide variety of subtle allelic alterations including additions and deletions of 1～18 bp in length in ZFN-injected samples. Most importantly, one of the 4 adults examined was capable of germline transmission to 15.2 % of its F1 progeny. Interestingly, ontogenic analyses of the allelic profile revealed that GD commenced early in development, continued during subsequent stages of development and in primordia for different adult organs of the three germ layers. These results demonstrate the feasibility and—for the first time to our knowledge—the efficacy of ZFN-mediated direct GD on a chromosomal gene in medaka embryos.     

An investigation of biomarkers derived from legacy microarray data for their utility in the RNA-seq era

Background

Gene expression microarray has been the primary biomarker platform ubiquitously applied in biomedical research, resulting in enormous data, predictive models, and biomarkers accrued. Recently, RNA-seq has looked likely to replace microarrays, but there will be a period where both technologies co-exist. This raises two important questions: Can microarray-based models and biomarkers be directly applied to RNA-seq data? Can future RNA-seq-based predictive models and biomarkers be applied to microarray data to leverage past investment?

Results

We systematically evaluated the transferability of predictive models and signature genes between microarray and RNA-seq using two large clinical data sets. The complexity of cross-platform sequence correspondence was considered in the analysis and examined using three human and two rat data sets, and three levels of mapping complexity were revealed. Three algorithms representing different modeling complexity were applied to the three levels of mappings for each of the eight binary endpoints and Cox regression was used to model survival times with expression data. In total, 240,096 predictive models were examined.

Conclusions

Signature genes of predictive models are reciprocally transferable between microarray and RNA-seq data for model development, and microarray-based models can accurately predict RNA-seq-profiled samples; while RNA-seq-based models are less accurate in predicting microarray-profiled samples and are affected both by the choice of modeling algorithm and the gene mapping complexity. The results suggest continued usefulness of legacy microarray data and established microarray biomarkers and predictive models in the forthcoming RNA-seq era.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0523-y) contains supplementary material, which is available to authorized users.     

Determination of zeranol and its metabolites in bovine muscle and liver by a chemiluminescence enzyme immunoassay: compared to an ultraperformance liquid chromatography tandem mass spectroscopy method

A chemiluminescent enzyme immunoassay (CLEIA) was compared to an ultraperformance liquid chromatography tandem mass spectroscopy (UPLC‐MS/MS) procedure for the analysis of zeranol and its metabolites in bovine tissue samples. Apparent recoveries from fortified samples by both methods were comparable at 0.5–4.0 µg/kg and a significant correlation was obtained. For CLEIA analysis, hapten mimicking the analyte was first synthesized and conjugated with the carrier protein bovine serum albumin as the immunogen to produce monoclonal antibody. The obtained antibody showed extensive cross‐reactivity toward zeranol metabolites (zearalanone). The limit of detection of CLEIA and UPLC‐MS/MS was 0.05 µg/kg and 0.5 µg/kg, respectively. Recoveries of both methods for fortified samples were higher than 75.0% with the coefficient of variation less than 15%. These results indicated that the combination of screening with CLEIA and confirmation with UPLC‐MS/MS for zeranol and its metabolites would be a reliable method for a large number of bovine samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Molecular photoacoustic tomography of breast cancer using receptor targeted magnetic iron oxide nanoparticles as contrast agents

In this report, we present a breast imaging technique combining high‐resolution near‐infrared (NIR) light induced photoacoustic tomography (PAT) with NIR dye‐labeled amino‐terminal fragments of urokinase plasminogen activator receptor (uPAR) targeted magnetic iron oxide nanoparticles (NIR830‐ATF‐IONP) for breast cancer imaging using an orthotopic mouse mammary tumor model. We show that accumulation of the targeted nanoparticles in the tumor led to photoacoustic contrast enhancement due to the high absorption of iron oxide nanoparticles (IONP). NIR fluorescence images were used to validate specific delivery of NIR830‐ATF‐IONP to mouse mammary tumors. We found that systemic delivery of the targeted IONP produced 4‐ and 10‐fold enhancement in photoacoustic signals in the tumor, compared to the tumor of the mice that received non‐targeted IONP or control mice. The use of targeted nanoparticles allowed imaging of tumors located as deep as 3.1 cm beneath the normal tissues. Our study indicates the potential of the combination of photoacoustic tomography and receptor‐targeted NIR830‐ATF‐IONP as a clinical tool that can provide improved specificity and sensitivity for breast cancer detection. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Improvement and Optimization of Standards for a Preclinical Animal Test Model of Laser Induced Choroidal Neovascularization

Background

As the murine model of laser-induced choroidal neovascularization (CNV) is becoming the most established and commonly utilized model worldwide for studying the pathogenesis of CNV and its response to treatment, specific operating standards are yet to be clarified. The purpose of this study is to compare the lesion size of CNV in mice with different ages, sex, durations of CNV process, and treated positions of laser spots, to make recommendations that may improve and optimize the quality of the model.

Methods and Results

C57/BL6 mice of different ages were treated with diode laser photocoagulation per eye and perfused with PBS containing fluorescein-labeled dextran at different time of observation. Choroid flat mounts, were then examined by fluorescence microscopy for the measurement of CNV area. Messenger-RNA expression levels of several angiogenic cytokines in eye cups of male and female C57BL/6 mice at 5–8 and 16–20 week-old were analyzed by real-time RT-PCR assay. The results showed significantly more CNV area in eyes of female mice compared to male mice with the expression level of several angiogenic cytokines elevated. 16–20-week-old female mice developed the biggest area of CNV. The mean area of CNV increased significantly at the 14^th day after photocoagulation. Laser spots delivered 1PD away from the optic disc induced the biggest area of CNV compared to those 2PD or 3PD away. Interaction of NV was observed in laser spots delivered less than 1PD away from each other.

Conclusion

The current results suggest that 16–20-week-old female C57BL/6 mice developed the most distinct CNV lesion size with laser spots delivered 1PD away from the optic disc. The best time to observe and analyze is the 14^th day after photocoagulation.     

Changes in the Expression of the Toll-Like Receptor System in the Aging Rat Kidneys

Background

The mechanisms of kidney aging are not yet clear. Studies have shown that immunological inflammation is related to kidney aging. Toll-like receptors (TLRs) are one of the receptor types of the body''s innate immune system. The function of the TLR system and the mechanisms by which it functions in renal aging remain unclear. In the present study, we, for the first time, systematically investigated the role of the TLR system and the inflammation responses activated by TLRs during kidney aging.

Methods

We used western blot and immunohistochemistry to systematically analyze the changes in the expression and activation of the endogenous TLR ligands HSP70 and HMGB1, the TLRs (TLR1–TLR11), their downstream signaling pathway molecules MyD88 and Phospho-IRF-3, and the NF-κB signaling pathway molecules Phospho-IKKβ, Phospho-IκBα (NF-κB inhibition factor α), NF-κBp65, and Phospho-NF-κBp65 (activated NF-κB p65) in the kidneys of 3 months old (youth group), 12 months old (middle age group), and 24 months old (elderly group) rats. We used RT-qPCR to detect the mRNA expression changes of the proinflammatory cytokines CCL3, CCL4, CCL5, CD80, TNF-α, and IL-12b in the rat renal tissues of the various age groups.

Results

We found that during kidney aging, the HSP70 and HMGB1 expression levels were significantly increased, and the expression levels of TLR1, 2, 3, 4, 5, and 11 and their downstream signaling pathway molecules MyD88 and Phospho-IRF-3 were markedly elevated. Further studies have shown that in the aging kidneys, the expression levels of the NF-κB signaling pathway molecules Phospho-IKKβ, Phospho-IκBα, NF-κBp65, and Phospho-NF-κBp65 were obviously increased, and those of the proinflammatory cytokines CCL3, CCL4, CCL5, CD80, TNF-α, and IL-12b were significantly upregulated.

Conclusions

These results showed that the TLR system might play an important role during the kidney aging process maybe by activating the NF-κB signaling pathway and promoting the high expression of inflammation factors.     

A Two-Stage Association Study Suggests BRAP as a Susceptibility Gene for Schizophrenia

Schizophrenia (SZ) is a neurodevelopmental disorder in which altered immune function typically plays an important role in mediating the effect of environmental insults and regulation of inflammation. The breast cancer suppressor protein associated protein (BRAP) is suggested to exert vital effects in neurodevelopment by modulating the mitogen-activated protein kinase cascade and inflammation signaling. To explore the possible role of BRAP in SZ, we conducted a two-stage study to examine the association of BRAP polymorphisms with SZ in the Han Chinese population. In stage one, we screened SNPs in BRAP from our GWAS data, which detected three associated SNPs, with rs3782886 being the most significant one (P  =  2.31E-6, OR  =  0.67). In stage two, we validated these three SNPs in an independently collected population including 1957 patients and 1509 controls, supporting the association of rs3782886 with SZ (P  =  1.43E-6, OR  =  0.73). Furthermore, cis-eQTL analysis indicates that rs3782886 genotypes are associated with mRNA levels of aldehyde dehydrogenase 2 family (ALDH2) (P  =  0.0039) and myosin regulatory light chain 2 (MYL2) (P < 1.0E-4). Our data suggest that the BRAP gene may confer vulnerability for SZ in Han Chinese population, adding further evidence for the involvement of developmental and/or neuroinflammatory cascades in the illness.