Promoter keyholes enable specific and persistent multi-gene expression programs in primary T cells without genome modification

Non-invasive epigenome editing is a promising strategy for engineering gene expression programs, yet potency, specificity, and persistence remain challenging. Here we show that effective epigenome editing is gated at single-base precision via 'keyhole' sites in endogenous regulatory DNA. Synthetic repressors targeting promoter keyholes can ablate gene expression in up to 99% of primary cells with single-gene specificity and can seamlessly repress multiple genes in combination. Transient exposure of primary T cells to keyhole repressors confers mitotically heritable silencing that persists to the limit of primary cultures in vitro and for at least 4 weeks in vivo, enabling manufacturing of cell products with enhanced therapeutic efficacy. DNA recognition and effector domains can be encoded as separate proteins that reassemble at keyhole sites and function with the same efficiency as single chain effectors, enabling gated control and rapid screening for novel functional domains that modulate endogenous gene expression patterns. Our results provide a powerful and exponentially flexible system for programming gene expression and therapeutic cell products

De novo design of protein logic gates

The design of modular protein logic for regulating protein function at the posttranscriptional level is a challenge for synthetic biology. Here, we describe the design of two-input AND, OR, NAND, NOR, XNOR, and NOT gates built from de novo–designed proteins. These gates regulate the association of arbitrary protein units ranging from split enzymes to transcriptional machinery in vitro, in yeast and in primary human T cells, where they control the expression of the TIM3 gene related to T cell exhaustion. Designed binding interaction cooperativity, confirmed by native mass spectrometry, makes the gates largely insensitive to stoichiometric imbalances in the inputs, and the modularity of the approach enables ready extension to three-input OR, AND, and disjunctive normal form gates. The modularity and cooperativity of the control elements, coupled with the ability to de novo design an essentially unlimited number of protein components, should enable the design of sophisticated posttranslational control logic over a wide range of biological functions

Promoter keyholes enable specific and persistent multi-gene expression programs in primary T cells without genome modification

Non-invasive epigenome editing is a promising strategy for engineering gene expression programs, yet potency, specificity, and persistence remain challenging. Here we show that effective epigenome editing is gated at single-base precision via 'keyhole' sites in endogenous regulatory DNA. Synthetic repressors targeting promoter keyholes can ablate gene expression in up to 99% of primary cells with single-gene specificity and can seamlessly repress multiple genes in combination. Transient exposure of primary T cells to keyhole repressors confers mitotically heritable silencing that persists to the limit of primary cultures in vitro and for at least 4 weeks in vivo, enabling manufacturing of cell products with enhanced therapeutic efficacy. DNA recognition and effector domains can be encoded as separate proteins that reassemble at keyhole sites and function with the same efficiency as single chain effectors, enabling gated control and rapid screening for novel functional domains that modulate endogenous gene expression patterns. Our results provide a powerful and exponentially flexible system for programming gene expression and therapeutic cell products

Comparison of methods for proanthocyanidin extraction from pine (Pinus densiflora) needles and biological activities of the extracts

Flavonoids are known to be effective scavengers of free radicals. In particular, proanthocyanidins are flavonoids that possess cardiovascular protection, antioxidative activities, and immunomodulatory activities. Here, we evaluated proanthocyanidin contents in the total polyphenolic compounds of pine needle extracts prepared by hot water, ethanol, hexane, hot water-hexane (HWH), and hot water-ethanol (HWE). Analysis of each extract indicated that the ethanol extract contained the highest proanthocyanidin concentration. The HWH and hexane extracts also contained relatively high concentrations of proanthocyanidin. On the other hand, proanthocyanidin content analyses out of the total polyphenolic compounds indicated that the HWH extract contained the highest content. These results suggest that HWH extraction is a suitable method to obtain an extract with a high level of pure proanthocyanidins and a relatively high yield. The HWH extract possessed superior activity in diverse antioxidative analyses such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferrous ion chelating (FIC), and ferric-ion reducing power (FRAP) assays. In addition, upon assessing the effects of the pine needle extracts on macrophages (Raw 264.7 cell), the HWH extract exhibited the highest activity. In this study, we discerned an efficient extraction method to achieve relatively pure proanthocyanidins from pine needles and evaluated the biological functions of the resulting extract, which could potentially be used for its efficacious components in functional food products

Graphene Schottky diodes: an experimental review of the rectifying graphene/semiconductor heterojunction

In the past decade graphene has been one of the most studied material for several unique and excellent properties. Due to its two dimensional nature, physical and chemical properties and ease of manipulation, graphene offers the possibility of integration with the exiting semiconductor technology for next-generation electronic and sensing devices. In this context, the understanding of the graphene/semiconductor interface is of great importance since it can constitute a versatile standalone device as well as the building-block of more advanced electronic systems. Since graphene was brought to the attention of the scientific community in 2004, the device research has been focused on the more complex graphene transistors, while the graphene/semiconductor junction, despite its importance, has started to be the subject of systematic investigation only recently. As a result, a thorough understanding of the physics and the potentialities of this device is still missing. The studies of the past few years have demonstrated that graphene can form junctions with 3D or 2D semiconducting materials which have rectifying characteristics and behave as excellent Schottky diodes. The main novelty of these devices is the tunable Schottky barrier height, a feature which makes the graphene/semiconductor junction a great platform for the study of interface transport mechanisms as well as for applications in photo-detection, high-speed communications, solar cells, chemical and biological sensing, etc. In this paper, we review the state-of-the art of the research on graphene/semiconductor junctions, the attempts towards a modeling and the most promising applications.Comment: 85 pages. Review articl

Analyst information precision and small earnings surprises

This study proposes and tests an alternative to the extant earnings management explanation for zero and small positive earnings surprises (i.e., analyst forecast errors). We argue that analysts’ ability to strategically induce slight pessimism in earnings forecasts varies with the precision of their information. Accordingly, we predict that the probability that a firm reports a small positive instead of a small negative earnings surprise is negatively related to earnings forecast uncertainty, and we present evidence consistent with this prediction. Our findings have important implications for the earnings management interpretation of the asymmetry around zero in the frequency distribution of earnings surprises. We demonstrate how empirically controlling for earnings forecast uncertainty can materially change inferences in studies that employ the incidence of zero and small positive earnings surprises to categorize firms as suspected of managing earnings

The cooperation of PIK3CA[H1047R] with BRAF[V600E] and KRAS[G12D] in lung tumorigenesis

KRAS is a GTPase that transmits external signals into the cell to regulate numerous cellular processes including cell division and apoptosis. Hence mutations in KRAS that render it constitutively active make it a potent oncogene. KRAS driver mutations are found in ~30% of non-small cell lung cancers and are associated with poor prognosis. Because of the significant challenges presented by pharmacological targeting of KRAS, efforts have turned to defining and targeting KRAS-regulated signaling pathways that are required for cancer cell maintenance. Most notable among these are the RAF protein kinases and PI3Kα, which have been credentialed both as key effectors of KRAS and bona fide human oncogenes. To study the contributions of these pathways to lung tumorigenesis I employed genetically engineered mouse models in which mutationally activated KRAS[G12D], BRAF[V600E], or PI3Kα[H1047R] can be conditionally expressed in the lung epithelium. Using these tools and complimentary tissue culture systems I tested the role of PI3Kα signaling in BRAF- and KRAS-driven lung cancer formation. My results show that simultaneous expression of oncogenic BRAF and PI3Kα is sufficient to form lung cancer in mice in a way that mimics KRAS activation. Thus RAF>MEK>ERK and PI3Kα>AKT act synergistically to promote lung tumorigenesis. In addition, mutational activation of PI3Kα dramatically accelerates KRAS-driven lung tumor initiation and progression, showing that despite the known ability of KRAS to directly activate PI3Kα, PI3-lipid signaling remains limiting for KRAS-driven lung tumorigenesis. Finally, the cooperation between oncogenic PI3Kα and BRAF or KRAS can be partially explained by signals downstream of RAF>MEK>ERK and PI3Kα>AKT that converge on activation of the cell division cycle and suppression of apoptosis. However in both BRAF/PIK3CA and KRAS/PIK3CA mutated tumors, PI3K inhibition is cytostatic at best and only moderately slows the growth of cancers, indicating that blockade of this pathway in established tumors suppresses cell division but does not illicit cell death

Comparative study of copper surface treatment with self-assembled monolayers of aliphatic thiol, dithiol and dithiocarboxylic acid

The stability of alkanethiol self-assembled monolayers (SAMs) on metallic substrates, in particular copper, is an important aspect for their application. Some technologies, such as lithographic patterning, can potentially benefit from the use of a transient easily removal protective coatings. Other applications like corrosion resistance, and thin-film lubrication request rather robust and stable films. The aim of this work consists in a comparative investigation of three organothiols (n-tetradecanethiol or RSH, 2-dodecylpropane-1,3-dithiol or R(SH)2 and n-tetradecanedithiocarboxylic or RS2H) on their ability to form SAMs on electrochemically reduced polycrystalline copper substrates and their respective stability. Characterizations of the SAMs are carried out using contact angle goniometry, XPS, PM-IRRAS and electrochemical studies (CV, LSV and cathodic desorption). R(SH)2 leads to monolayers with lower molecular organization than in the case of RSH and RS2H molecules. R(SH)2 monolayers are found the more stable when subjected to both anodic and cathodic desorption tests. © 2009 Elsevier B.V. All rights reserved