Investigating the functionality of an OCT4-short response element in human induced pluripotent stem cells.

Pluripotent stem cells offer great therapeutic promise for personalized treatment platforms for numerous injuries, disorders, and diseases. Octamer-binding transcription factor 4 (OCT4) is a key regulatory gene maintaining pluripotency and self-renewal of mammalian cells. With site-specific integration for gene correction in cellular therapeutics, use of the OCT4 promoter may have advantages when expressing a suicide gene if pluripotency remains. However, the human OCT4 promoter region is 4 kb in size, limiting the capacity of therapeutic genes and other regulatory components for viral vectors, and decreasing the efficiency of homologous recombination. The purpose of this investigation was to characterize the functionality of a novel 967bp OCT4-short response element during pluripotency and to examine the OCT4 titer-dependent response during differentiation to human derivatives not expressing OCT4. Our findings demonstrate that the OCT4-short response element is active in pluripotency and this activity is in high correlation with transgene expression in vitro, and the OCT4-short response element is inactivated when pluripotent cells differentiate. These studies demonstrate that this shortened OCT4 regulatory element is functional and may be useful as part of an optimized safety component in a site-specific gene transferring system that could be used as an efficient and clinically applicable safety platform for gene transfer in cellular therapeutics

Ancient Maya Regional Settlement and Inter-Site Analysis: The 2013 West-Central Belize LiDAR Survey

During April and May 2013, a total of 1057 km2 of LiDAR was flown by NCALM for a consortium of archaeologists working in West-central Belize, making this the largest surveyed area within the Mayan lowlands. Encompassing the Belize Valley and the Vaca Plateau, West-central Belize is one of the most actively researched parts of the Maya lowlands; however, until this effort, no comprehensive survey connecting all settlement had been conducted. Archaeological projects have investigated at least 18 different sites within this region. Thus, a large body of archaeological research provides both the temporal and spatial parameters for the varied ancient Maya centers that once occupied this area; importantly, these data can be used to help interpret the collected LiDAR data. The goal of the 2013 LiDAR campaign was to gain information on the distribution of ancient Maya settlement and sites on the landscape and, particularly, to determine how the landscape was used between known centers. The data that were acquired through the 2013 LiDAR campaign have significance for interpreting both the composition and limits of ancient Maya political units. This paper presents the initial results of these new data and suggests a developmental model for ancient Maya polities

Modulation of Cell Adhesion and Migration by the Histone Methyltransferase Subunit mDpy-30 and Its Interacting Proteins

We have previously shown that a subset of mDpy-30, an accessory subunit of the nuclear histone H3 lysine 4 methyltransferase (H3K4MT) complex, also localizes at the trans-Golgi network (TGN), where its recruitment is mediated by the TGN-localized ARF guanine nucleotide exchange factor (ArfGEF) BIG1. Depletion of mDpy-30 inhibits the endosome-to-TGN transport of internalized CIMPR receptors and concurrently promotes their accumulation at the cell protrusion. These observations suggest mDpy-30 may play a novel role at the crossroads of endosomal trafficking, nuclear transcription and adhesion/migration. Here we provide novel mechanistic and functional insight into this association. First, we demonstrate a direct interaction between mDpy-30 and BIG1 and locate the binding region in the N-terminus of BIG1. Second, we provide evidence that the depletion or overexpression of mDpy-30 enhances or inhibits cellular adhesion/migration of glioma cells in vitro, respectively. A similar increase in cell adhesion/migration is observed in cells with reduced levels of BIG1 or other H3K4MT subunits. Third, knockdown of mDpy-30, BIG1, or the RbBP5 H3K4MT subunit increases the targeting of β1 integrin to cell protrusions, and suppression of H3K4MT activity by depleting mDpy-30 or RbBP5 leads to increased protein and mRNA levels of β1 integrin. Moreover, stimulation of cell adhesion/migration via mDpy-30 knockdown is abolished after treating cells with a function-blocking antibody to β1 integrin. Taken together, these data indicate that mDpy-30 and its interacting proteins function as a novel class of cellular adhesion/migration modulators partially by affecting the subcellular distribution of endosomal compartments as well as the expression of key adhesion/migration proteins such as β1 integrin

Ancient Lowland Maya neighborhoods: Average Nearest Neighbor analysis and kernel density models, environments, and urban scale

Many humans live in large, complex political centers, composed of multi-scalar communities including neighborhoods and districts. Both today and in the past, neighborhoods form a fundamental part of cities and are defined by their spatial, architectural, and material elements. Neighborhoods existed in ancient centers of various scales, and multiple methods have been employed to identify ancient neighborhoods in archaeological contexts. However, the use of different methods for neighborhood identification within the same spatiotemporal setting results in challenges for comparisons within and between ancient societies. Here, we focus on using a single method—combining Average Nearest Neighbor (ANN) and Kernel Density (KD) analyses of household groups—to identify potential neighborhoods based on clusters of households at 23 ancient centers across the Maya Lowlands. While a one-size-fits all model does not work for neighborhood identification everywhere, the ANN/KD method provides quantifiable data on the clustering of ancient households, which can be linked to environmental zones and urban scale. We found that centers in river valleys exhibited greater household clustering compared to centers in upland and escarpment environments. Settlement patterns on flat plains were more dispersed, with little discrete spatial clustering of households. Furthermore, we categorized the ancient Maya centers into discrete urban scales, finding that larger centers had greater variation in household spacing compared to medium-sized and smaller centers. Many larger political centers possess heterogeneity in household clustering between their civic-ceremonial cores, immediate hinterlands, and far peripheries. Smaller centers exhibit greater household clustering compared to larger ones. This paper quantitatively assesses household clustering among nearly two dozen centers across the Maya Lowlands, linking environment and urban scale to settlement patterns. The findings are applicable to ancient societies and modern cities alike; understanding how humans form multi-scalar social groupings, such as neighborhoods, is fundamental to human experience and social organization

Late Cretaceous UHP metamorphism recorded in kyanite-garnet schists from the Central Rhodope Mountains, Bulgaria.

In this study, we report the first discovery of microdiamond inclusions in kyanite–garnet schists from the Central Rhodope Mountains in Bulgaria. These inclusions occur in garnets from metapelites that are part of a meta-igneous and meta-sedimentary mélange hosted by Variscan (Hercynian) orthogneiss. Ultra-high-pressure (UHP) conditions are further supported by the presence of exsolved needles of quartz and rutile in the garnet and by geothermobarometry estimates that suggest peak metamorphic temperatures of 750–800 °C and pressures in excess of 4 GPa. The discovery of UHP conditions in the Central Rhodopes of Bulgaria compliments the well-documented evidence for such conditions in the southernmost (Greek) part of the Rhodope Massif. Dating of garnets from these UHP metapelites (Chepelare Shear Zone) using Sm–Nd geochronology indicates a Late Cretaceous age (70.5–92.7 Ma) for the UHP metamorphic event. This is significantly younger than previously reported ages and suggests that the UHP conditions are associated with the Late Mesozoic subduction of the Vardar Ocean northward beneath the Moesian platform (Europe). The present-day structure of the RM is the result of a series of subduction–exhumation events that span the Cenozoic, alongside subsequent post-orogenic extension and metamorphic core complex formation

Investigating the Mechanisms of Reprogramming and Optimizing the Generation of Potentially Therapeutically Useful Induced Pluripotent Stem Cell Derivatives

Human induced pluripotent stem cells (hiPSCs), derived from easily obtainable skin cells, possess enormous opportunity for autologous cellular treatment therapies, gene correction, and disease modeling without worries of ethical constraints associated with human embryonic stem cells (hESCs). Although lentiviral based reprogramming remains as one of the most popular methods for reprogramming, potentially oncogenic viral integrations in random locations throughout the genome along with non-human antigens associated with the reprogramming process thwart the clinical applications of these hiPSCs. To address these concerns we derived a hiPSC line void of any exogenous reprogramming factors and differentiated these hiPSCs into clinically relevant cell derivatives. In addition, to maintain clinical relevance, we implemented a methodology to clean our hiPSCs from non-human antigens to allow for current good manufacturing practice conditions that could help set the standard for human clinical trials with our factor-free hiPSCs. The field of stem cell reprogramming has rapidly advanced, and a new technique involving mRNA based reprogramming was introduced that we found to be difficult to reproduce due to an innate immune response based degradation of mRNA when introduced into the cell. To solve this problem, a small chemical compound was utilized that blocked important aspects of the innate immune response to single stranded mRNA that yielded robust and uniform expression of a key reprogramming factor. This stabilization could be important in increasing mRNA based reprogramming efficiency of hiPSC derivation. Another challenge in the hiPSC field is investigating nuanced potential differences manifested in transcriptional, epigenetic, immunological, and differentiation potentials between hESCs and hiPSCs. To help and potentially solve this problem and allow for more complete and faithful reprogramming to a hESC state, global microarray transcriptional analysis of oocyte cytoplasm was utilized to find eight putative novel shared reprogramming factors across multiple species. These factors have identifiable roles in opening up chromatin that can allow reprogramming factors to better access reprogramming loci that could confer the known reprogramming advantage that somatic cell nuclear transfer based reprogramming maintains over current direct reprogramming approaches. To address the recently observed immunogenicity issues of iPSCs, we studied the expression of two normally fetally associated genes implicated in an iPSC-specific immune response. We found high line-to-line variation between both hESC and hiPSC lines across different levels of differentiation and confirmed that current differentiation protocols derive cell types with a fetal phenotype as opposed to the adult phenotype needed for clinical applications as indicated by aberrant expression of specific fetal genes. Taken altogether, we hope these studies allow for more robust, reproducible, and clinically relevant hiPSCs that more closely resemble hESCs and maintain full ability to differentiate into clinically relevant cell types that can be used for potential human clinical trials for disease and cell replacement therapy

BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells

Abstract Introduction The OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. Synthetic OCT4 mRNA was recently used (in conjunction with other reprogramming factors) to generate human induced pluripotent stem cells. Here, we discovered that BAY 11-7082 (BAY11), at least partially through an NF-κB-inhibition based mechanism, could significantly increase the expression of OCT4 following transfection of synthetic mRNA (synRNA) into adult human skin cells. Methods We tested various chemical and molecular small molecules on their ability to suppress the innate immune response seen upon synthetic mRNA transfection. Three molecules - B18R, BX795, and BAY11 - were used in immunocytochemical and proliferation-based assays. We also utilized global transcriptional meta-analysis coupled with quantitative PCR to identify relative gene expression downstream of OCT4. Results We found that human skin cells cultured in the presence of BAY11 resulted in reproducible increased expression of OCT4 that did not inhibit normal cell proliferation. The increased levels of OCT4 resulted in significantly increased expression of genes downstream of OCT4, including the previously identified SPP1, DUSP4 and GADD45G, suggesting the expressed OCT4 was functional. We also discovered a novel OCT4 putative downstream target gene SLC16A9 which demonstrated significantly increased expression following elevation of OCT4 levels. Conclusions For the first time we have shown that small molecule-based stabilization of synthetic mRNA expression can be achieved with use of BAY11. This small molecule-based inhibition of innate immune responses and subsequent robust expression of transfected synthetic mRNAs may have multiple applications for future cell-based research and therapeutics