Determining glioblastoma proteome changes in response to lateral ventricle neural stem cells

Glioblastoma (GBM) is the most common and malignant primary tumor in adults. When GBM tumors are located close to the lateral ventricle they display a more aggressive recurrence pattern and negatively impact patient survival. These findings suggest the involvement of the subventricular zone neurogenic niche in GBM malignancy. To define the inter-cellular communication between neural stem cells and GBM cells, we optimized a tool to determine cell-specific proteomic changes of GBM cells in response to neural stem cell proximity. We cloned the mutated methionyl-tRNA synthetase (MetRS) gene into the lentiviral plasmid MetRS puro. MetRS allows for incorporation of azide-tagged methionine analog azidonorleucine (ANL) into newly formed proteins, effectively labeling proteins synthesized by expressing cells. We utilized the pLKO.1 vector backbone allowing puromycin resistance as a selection method. The MetRS L274 modification was confirmed, as only MetRS-transduced cells of both commercial HEK and primary GBM1A cell lines selectively incorporated ANL. Following verification, we successfully packaged the plasmid into a lentivirus. We transduced primary human fetal neural stem cell (hfNSC) and GBM lines and selected the MetRS-expressing cells by puromycin exposure. After 96 hours, wild type (WT) cells died while successfully transduced cells exhibited resistance and the ANL-compatible MetRS enzyme. Co-cultures consisting of MetRS-transduced GBM and WT hfNSCs were used to simulate a similar environment of glioblastoma neighboring lateral ventricles. Proteome Profiler results showed a significant downregulation of an angiogenesis inhibitor and upregulation of malignancy promoting proteins in GBM1A. Going forward, this analysis method will be used for cell-specific proteomics in vivo

ASASSN-15lu is a Type Ia Supernova

We report spectroscopic classification of ASASSN-15lu (ATel #7698) in SDSS J132112.88+401556.7 (z=0.035037, via NED) through inspection of an optical spectrum (range 370-680 nm, resolution 0.8 nm), obtained with the 2.3-m Bok telescope (+ Boller & Chivens spectrograph) at Kitt Peak on 2015 June 24.2 UT

Animal welfare during transport and slaughter of beef cattle

Objective: To review the way transport and stun cattle affect animal welfare. Approach: During the transport of cattle to slaughter plants, many factors affect animal welfare, such as travel time, stress and load density. Besides, stunning might comply with animal welfare guidelines, which different protocols such as Welfare Quality® establish.Objective: To review how transport and stunning of cattle affect animal welfare. Approach: During the transport of beef cattle to slaughter plants, several factors affect animal welfare, such as travel time, stress, and load density. Additionally, the correct stunning of cattle helps comply with the animal welfare guidelines established by different protocols such as Welfare Quality®. Study limitations/Implications: Meat quality is affected by several factors, being of utmost importance the way animals are transported to the slaughterhouse, and they are stunned. Therefore, it is critical to perform these stages properly to obtain good quality meat; besides, it is a welfare issue. Conclusions: It is critical to comply with transport and slaughter procedures that guarantee good beef meat quality and ensure animal welfare to avoid stress in cattle as possible

Sub-population analysis based on temporal features of high content images

Background: High content screening techniques are increasingly used to understand the regulation and progression of cell motility. The demand of new platforms, coupled with availability of terabytes of data has challenged the traditional technique of identifying cell populations by manual methods and resulted in development of high-dimensional analytical methods. Results: In this paper, we present sub-populations analysis of cells at the tissue level by using dynamic features of the cells. We used active contour without edges for segmentation of cells, which preserves the cell morphology, and autoregressive modeling to model cell trajectories. The sub-populations were obtained by clustering static, dynamic and a combination of both features. We were able to identify three unique sub-populations in combined clustering. Conclusion: We report a novel method to identify sub-populations using kinetic features and demonstrate that these features improve sub-population analysis at the tissue level. These advances will facilitate the application of high content screening data analysis to new and complex biological problems.Computation and Systems Biology Programme of Singapore--Massachusetts Institute of Technology Allianc

MicroRNA-520b Inhibits Growth of Hepatoma Cells by Targeting MEKK2 and Cyclin D1

Growing evidence indicates that the deregulation of microRNAs (miRNAs) contributes to the tumorigenesis. We previously revealed that microRNA-520b (miR-520b) was involved in the complement attack and migration of breast cancer cells. In this report, we show that miR-520b is an important miRNA in the development of hepatocellular carcinoma (HCC). Our data showed that the expression levels of miR-520b were significantly reduced in clinical HCC tissues and hepatoma cell lines. We observed that the introduction of miR-520b dramatically suppressed the growth of hepatoma cells by colony formation assays, 5-ethynyl-2-deoxyuridine (EdU) incorporation assays and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Moreover, ectopic expression of miR-520b was able to inhibit the growth of hepatoma cells in nude mice. Further studies revealed that the mitogen-activated protein kinase kinase kinase 2 (MEKK2) and cyclin D1 were two of direct target genes of miR-520b. Silencing of MEKK2 or cyclin D1 was able to inhibit the growth of hepatoma cells in vitro and in vivo, which is consistent with the effect of miR-520b overexpression on the growth of hepatoma cells. In addition, miR-520b significantly decreased the phosphorylation levels of c-Jun N-terminal kinase (p-JNK, a downstream effector of MEKK2) or retinoblastoma (p-Rb, a downstream effector of cyclin D1). In conclusion, miR-520b is able to inhibit the growth of hepatoma cells by targeting MEKK2 or cyclin D1 in vitro and in vivo. Our findings provide new insights into the role of miR-520b in the development of HCC, and implicate the potential application of miR-520b in cancer therapy

Interactions between cytoplasmic and nuclear genomes confer sex‐specific effects on lifespan in Drosophila melanogaster

Genetic variation outside of the cell nucleus can affect the phenotype. The cytoplasm is home to the mitochondria, and in arthropods often hosts intracellular bacteria such as Wolbachia. Although numerous studies have implicated epistatic interactions between cytoplasmic and nuclear genetic variation as mediators of phenotypic expression, two questions remain. Firstly, it remains unclear whether outcomes of cyto-nuclear interactions will manifest differently across the sexes, as might be predicted given that cytoplasmic genomes are screened by natural selection only through females as a consequence of their maternal inheritance. Secondly, the relative contribution of mitochondrial genetic variation to other cytoplasmic sources of variation, such as Wolbachia infection, in shaping phenotypic outcomes of cyto-nuclear interactions remains unknown. Here, we address these questions, creating a fully crossed set of replicated cyto-nuclear populations derived from three geographically distinct populations of Drosophila melanogaster, measuring the lifespan of males and females from each population. We observed that cyto-nuclear interactions shape lifespan and that the outcomes of these interactions differ across the sexes. Yet, we found no evidence that placing the cytoplasms from one population alongside the nuclear background of others (generating putative cyto-nuclear mismatches) leads to decreased lifespan in either sex. Although it was difficult to partition mitochondrial from Wolbachia effects, our results suggest at least some of the cytoplasmic genotypic contribution to lifespan was directly mediated by an effect of sequence variation in the mtDNA. Future work should explore the degree to which cyto-nuclear interactions result in sex differences in the expression of other components of organismal life history

Signal Transmission in the Auditory System

Contains table of contents for Section 3 and reports on four research projects.National Institutes of Health Grant R01 DC00194National Institutes of Health Grant P01 DC00119National Science Foundation Grant IBN 96-04642W.M. Keck Foundation Career Development ProfessorshipNational Institutes of Health Grant R01 DC00238Thomas and Gerd Perkins Award ProfessorshipAlfred P Sloan Foundation Instrumentation GrantJohn F. and Virginia B. Taplin Award in Health Sciences and TechnologyNational Institutes of Health/National Institute of Deafness and Other Communication DisordersNational Institutes of Health/National Institute of Deafness and Other Communication Disorders Grant PO1 DC0011

Multiple Statistical Analysis Techniques Corroborate Intratumor Heterogeneity in Imaging Mass Spectrometry Datasets of Myxofibrosarcoma

MALDI mass spectrometry can generate profiles that contain hundreds of biomolecular ions directly from tissue. Spatially-correlated analysis, MALDI imaging MS, can simultaneously reveal how each of these biomolecular ions varies in clinical tissue samples. The use of statistical data analysis tools to identify regions containing correlated mass spectrometry profiles is referred to as imaging MS-based molecular histology because of its ability to annotate tissues solely on the basis of the imaging MS data. Several reports have indicated that imaging MS-based molecular histology may be able to complement established histological and histochemical techniques by distinguishing between pathologies with overlapping/identical morphologies and revealing biomolecular intratumor heterogeneity. A data analysis pipeline that identifies regions of imaging MS datasets with correlated mass spectrometry profiles could lead to the development of novel methods for improved diagnosis (differentiating subgroups within distinct histological groups) and annotating the spatio-chemical makeup of tumors. Here it is demonstrated that highlighting the regions within imaging MS datasets whose mass spectrometry profiles were found to be correlated by five independent multivariate methods provides a consistently accurate summary of the spatio-chemical heterogeneity. The corroboration provided by using multiple multivariate methods, efficiently applied in an automated routine, provides assurance that the identified regions are indeed characterized by distinct mass spectrometry profiles, a crucial requirement for its development as a complementary histological tool. When simultaneously applied to imaging MS datasets from multiple patient samples of intermediate-grade myxofibrosarcoma, a heterogeneous soft tissue sarcoma, nodules with mass spectrometry profiles found to be distinct by five different multivariate methods were detected within morphologically identical regions of all patient tissue samples. To aid the further development of imaging MS based molecular histology as a complementary histological tool the Matlab code of the agreement analysis, instructions and a reduced dataset are included as supporting information