A functional screen for optimization of short hairpin RNA biogenesis and RISC loading

Gene silencing via short hairpin mediated RNAi (shRNA) is a valuable experimental tool and has promise as a therapeutic strategy. Several shRNA platforms make use of the loop and flanking sequences from the endogenous microRNA (miRNAs) miR-30a or other miRNAs to provide an RNA structure for efficient and accurate biogenesis of the RNA trigger. However, the stem regions of these shRNAs are typically designed as perfect duplex structures which is an uncommon feature for endogenous miRNA precursors. A limitation of these designs is that shRNAs with perfect duplex stems undergo extensive stem cleavage analogous to the Dicer independent miRNA miR-451, destroying the shRNA trigger sequence that is present in the 3P arm. We employed an unbiased screen of > 9000 shRNA structures to identify features that prevent stem cleavage and promote canonical biogenesis and loading into the effector complex RISC. We find that a central stem bulge or kink reduces central stem cleavage and improves accuracy of Dicer processing. Furthermore, 9 - 10 GC nucleotides in the guide strand improves shRNA efficiency. These design rules enable more effective shRNA tools and are compatible with existing sets of optimized guide/target sequences.

FloRunTM ‘331’ Peanut Variety

FloRunTM ‘331’ peanut variety was developed by the University of Florida, Institute of Food and Agricultural Sciences, North Florida Research and Education Center near Marianna, Florida. It was released in 2016 because it combines high yield potential with excellent disease tolerance. FloRunTM ‘331’ has a typical runner growth habit with a semi-prominent central stem and medium green foliage. It has medium runner seed size with high oleic oil chemistry.

Three branches of literary anthropology: Sources, styles, subject matter

‘What is literary anthropology?’ – a deceptively simple question, posed by anthropologist Paul Stoller – unleashes debate about the perceived identity of the field. Through the lens of three book reviews, this essay proposes conceptualizing literary anthropology as a central stem with three branches. The first is the use of literary texts as ethnographic source material, particularly for historical anthropologists. The second is the use of literary modes of writing ethnography, ranging from the incorporation of metaphorical language and the subversion of conventional ethnographic structures to the production of fiction as ethnography. The third is the anthropological examination of literary cultural and production practices. The third has been underexplored in the academy to date, the second has been at the centre of fierce controversy within the wider field of anthropology, while the first has arguably been limited by restrictive disciplinary and epistemological assumptions.

Optimal structure of heterogeneous stem cell niche: The importance of cell migration in delaying tumorigenesis

Studying the stem cell niche architecture is a crucial step for investigating the process of oncogenesis and obtaining an effective stem cell therapy for various cancers. Recently, it has been observed that there are two groups of stem cells in the stem cell niche collaborating with each other to maintain tissue homeostasis. One group comprises the border stem cells, which is responsible to control the number of non-stem cells as well as stem cells. The other group, central stem cells, regulates the stem cell niche. In the present study, we develop a bi-compartmental stochastic model for the stem cell niche to study the spread of mutants within the niche. The analytic calculations and numeric simulations, which are in perfect agreement, reveal that in order to delay the spread of mutants in the stem cell niche, a small but non-zero number of stem cell proliferations must occur in the central stem cell compartment. Moreover, the migration of border stem cells to the central stem cell compartment delays the spread of mutants. Furthermore, the fixation probability of mutants in the stem cell niche is independent of types of stem cell division as long as all stem cells do not divide fully asymmetrically. Additionally, the progeny of central stem cells have a much higher chance than the progeny of border stem cells to take over the entire niche.

The Novel Formation of Barium Titanate Nanodendrites

The barium titanate (BaTiO3) nanoparticles with novel dendrite-like structures have been successfully fabricated via a simple coprecipitation method, the so-called BaTiO3nanodendrites (BTNDs). This method was remarkable, fast, simple, and scalable. The growth solution is prepared by barium chloride (BaCl2), titanium tetrachloride (TiCl4), and oxalic acid. The shape and size of BaTiO3depend on the amount of added BaCl2solvent. To investigate the influence of amount of BaCl2on BTNDs, the amount of BaCl2was varied in the range from 3 to 6 mL. The role of BaCl2is found to have remarkable influence on the morphology, crystallite size, and formation of dendrite-like structures. The thickness and length of the central stem of BTND were ~300 nm and ~20 μm, respectively. The branchings were found to occur at irregular intervals along the main stem. Besides, the formation mechanism of BTND is proposed and discussed.

MODEL OF RAMIFICATION IN ELECTRODEPOSITED FRACTALS

The macroscopic branching and shape of a metal, electrodeposited in quasi-two dimensions, can be represented by a Lindenmayer System of multiple iterated function systems. The motif of a central branch that continues and two side branches is shown to allow modeling of the observed shapes. The dilation (or contraction) in length, and the angle of a branch with respect to the central stem are the only parameters needed to create realistic simulations of such ramified deposits. Values for these quantities together with standard deviations measured from a number of electrodeposited copper fractals have been used to generate simulations. Measurement of the parameters of the motif, the average linear dilation factor from one generation to the next, and the angles between branches, show a 5% relative standard deviation of these factors across one growth. The values of these parameters can also indicate the transition from an open fractal form to the more directed dendritic form. The results are compared with other approaches to describing these systems.

Identification and Mutational Analysis of a Rej Response Element in Jaagsiekte Sheep Retrovirus RNA

ABSTRACT Jaagsiekte sheep retrovirus (JSRV) is a simple betaretrovirus causing a contagious lung cancer of sheep. JSRV encodes unspliced and spliced viral RNAs, among which unspliced RNA encodes Gag and Pol proteins and a singly spliced mRNA encodes Env protein. In another study we found that JSRV encodes a regulatory protein, Rej, that is responsible for synthesis of Gag polyprotein from unspliced viral RNA. Rej is encoded in the 5′ end of env, and it enhances nuclear export or accumulation of cytoplasmic unspliced viral RNA in 293T cells but not in most other cell lines (A. Hofacre, T. Nitta, and H. Fan, J. Virol. 83:12483-12498, 2009). In this study, we found that mutations in the 3′ end of env in the context of a cytomegalovirus-driven full-length JSRV expression construct abolished Gag protein synthesis and released viruses in 293T cells. These mutants also showed deficits in accumulation of unspliced viral RNA in the cytoplasm. These mutants defined a Rej-responsive element (RejRE). Inhibition of CRM1 but not Tap function prevented nuclear export/accumulation of cytoplasmic unspliced RNA in 293T cells, similarly to other complex retroviruses that express analogous regulator proteins (e.g., human immunodeficiency virus Rev). Structural modeling of the RejRE with Zuker M-fold indicated a region with a predicted stable secondary structure. Mutational analysis in this region indicated the importance of both secondary structures and primary nucleotide sequences in a central stem-bulge-stem structure. In contrast to 293T cells, mutations in the RejRE did not affect the levels of cytoplasmic unspliced RNA in 293 cells, although the unspliced RNA showed partial degradation, perhaps due to lack of translation. RejRE-containing RNA relocalized Rej protein from the nucleus to the cytoplasm in 293 and rat 208F cells, suggesting binding of Rej to the RejRE.

Floral ontogeny of Swainsona formosa (Fabaceae: Faboideae: Galegeae)

Swainsona formosa (G.Don) J.Thompson (Sturt’s desert pea) is used in commercial floriculture for cut flowers and ornamental pot plants; however, accurate identification of the growth stages is critically important in making management decisions in floricultural crops. This plant was investigated by stereomicroscopy and scanning electron microscopy (SEM) to identify flowering time and stages of floral development. This is the first work to describe the complete floral ontogeny in a member of tribe Galegeae. Conversion from vegetative to reproductive stages began within 40–46 days after seed germination for axillary branches and within 46–52 days for central stems. Plants required 807.5 days °C growing degree-days for axillary branches and 921.5 days °C for central stems to reach 50% flowering. The central stem grew more nodes (11.1 ± 0.97 nodes) before the initiation of the first flower than did the axillary branches (7.2 ± 0.93 nodes). The order of floral organ initiation within each whorl is unidirectional, except for the petal whorl, which is simultaneous; the flower is organised into five whorls and shows a pentamerous arrangement of sepals and petals, 10 stamens in two whorls and a central carpel.

A Novel RNA Binding Domain in Tetrahymena Telomerase p65 Initiates Hierarchical Assembly of Telomerase Holoenzyme

ABSTRACT Telomerase reverse transcriptase (TERT) and telomerase RNA (TER) assemble as part of a holoenzyme that synthesizes telomeric repeats at chromosome ends. Genetic approaches have identified proteins that are required for in vivo association of TERT and TER, including the Tetrahymena telomerase holoenzyme protein p65. Here, we use quantitative assays to define the mechanisms underlying p65 function in holoenzyme biogenesis. We demonstrate that four modules of p65 contribute affinity for TER, including a C-terminal domain that recognizes the conserved dinucleotide bulge of central stem IV. This C-terminal domain is necessary and sufficient for p65's function in enhancing the recruitment of TERT to TER. Finally, we show that p65 and TERT assemble on TER with hierarchical rather than cooperative binding. These findings elucidate an extensive network of p65-TER recognition specificity and define a novel p65 RNA binding domain that initiates telomerase holoenyzme biogenesis.