RSEARCH: Finding homologs of single structured RNA sequences

BACKGROUND: For many RNA molecules, secondary structure rather than primary sequence is the evolutionarily conserved feature. No programs have yet been published that allow searching a sequence database for homologs of a single RNA molecule on the basis of secondary structure. RESULTS: We have developed a program, RSEARCH, that takes a single RNA sequence with its secondary structure and utilizes a local alignment algorithm to search a database for homologous RNAs. For this purpose, we have developed a series of base pair and single nucleotide substitution matrices for RNA sequences called RIBOSUM matrices. RSEARCH reports the statistical confidence for each hit as well as the structural alignment of the hit. We show several examples in which RSEARCH outperforms the primary sequence search programs BLAST and SSEARCH. The primary drawback of the program is that it is slow. The C code for RSEARCH is freely available from our lab's website. CONCLUSION: RSEARCH outperforms primary sequence programs in finding homologs of structured RNA sequences

Stratigraphy, age, and provenance of the Eocene Chumstick basin, Washington Cascades; implications for paleogeography, regional tectonics, and development of strike-slip basins: Reply

We welcome the comment by Evans (2022) and the opportunity to further discuss our study of the Chumstick Formation. The correlation of fault-bound nonmarine sedimentary units in central and western Washington has been a topic of interest, and debate, for many years (Frizzell, 1979; Taylor et al., 1988; Gresens et al., 1981; Gresens, 1983; Evans and Johnson, 1989; Evans, 1994; Cheney and Hayman, 2009). However, many questions about the regional correlation of these units were resolved with the publication of a suite of internally consistent high-precision 206Pb/238U zircon dates from volcanic interbeds throughout the early to middle Eocene stratigraphy (Eddy et al., 2016). This data set confirmed the timing of sediment deposition of the different members within the Chumstick Formation. Donaghy et al. (2021) provides a detailed study of the Chumstick Formation, which builds on earlier research by Gresens et al. (1981, 1983), McClincy (1986), and Evans (1994) by incorporating new geochronologic information and additional clast counts, detrital zircon geochronology, and facies mapping. We interpret large parts of the Chumstick Formation to represent a spatially and temporally distinct sedimentary system between the Leavenworth and Entiat fault zones that likely formed as a pull-apart basin. Evans (2022) objects to several of the interpretations presented in Donaghy et al. (2021) regarding the relationship between different members of the Chumstick Formation and surrounding sedimentary units, the timing of strike-slip faulting, and the regional tectonic setting of these rocks. We discuss each of these points in the following sections

MenaINV dysregulates cortactin phosphorylation to promote invadopodium maturation

Invadopodia, actin-based protrusions of invasive carcinoma cells that focally activate extracellular matrix-degrading proteases, are essential for the migration and intravasation of tumor cells during dissemination from the primary tumor. We have previously shown that cortactin phosphorylation at tyrosine residues, in particular tyrosine 421, promotes actin polymerization at newly-forming invadopodia, promoting their maturation to matrix-degrading structures. However, the mechanism by which cells regulate the cortactin tyrosine phosphorylation-dephosphorylation cycle at invadopodia is unknown. Mena, an actin barbed-end capping protein antagonist, is expressed as various splice-isoforms. The MenaINV isoform is upregulated in migratory and invasive sub-populations of breast carcinoma cells, and is involved in tumor cell intravasation. Here we show that forced MenaINV expression increases invadopodium maturation to a far greater extent than equivalent expression of other Mena isoforms. MenaINV is recruited to invadopodium precursors just after their initial assembly at the plasma membrane, and promotes the phosphorylation of cortactin tyrosine 421 at invadopodia. In addition, we show that cortactin phosphorylation at tyrosine 421 is suppressed by the phosphatase PTP1B, and that PTP1B localization to the invadopodium is reduced by MenaINV expression. We conclude that MenaINV promotes invadopodium maturation by inhibiting normal dephosphorylation of cortactin at tyrosine 421 by the phosphatase PTP1B.United States. National Institutes of Health (CA150344)United States. National Institutes of Health (CA100324

Eocene dike orientations across the Washington Cascades in response to a major strike-slip faulting episode and ridge-trench interaction

The northern Cascade Mountains in Washington (USA) preserve an exceptional shallow to mid-crustal record of Eocene transtension marked by dextral strike-slip faulting, intrusion of dike swarms and plutons, rapid non-marine sedimentation, and ductile flow and rapid cooling in parts of the North Cascades crystalline core. Transtension occurred during ridge-trench interaction with the formation of a slab window, and slab rollback and break-off occurred shortly after collision of the Siletzia oceanic plateau at ca. 50 Ma. Dike swarms intruded a \u3e1250 km2 region between ca. 49.3 Ma and 44.9 Ma, and orientations of more than 1500 measured dikes coupled with geochronologic data provide important snapshots of the regional strain field. The mafic Teanaway dikes are the southernmost and most voluminous of the swarms. They strike NE (mean = 036°) and average ~15 m in thickness. To the north, rhyolitic to basaltic dikes overlap spatially with 49.3-46.5 Ma, mainly granodioritic plutons, but they typically predate the nearby plutons by ca. 500 k.y. The average orientations of five of the six dike domains range from 010° to 058°; W-NW- to NW-striking dikes characterize one domain and are found in lesser amounts in a few other domains. Overall, the mean strike for all Eocene dikes is 035°, and the average extension direction (305°-125°) is oblique to the strike (~320°) of the North Cascades orogen. Extension by diking reached ~45% in one \u3e7-km-long transect through the Teanaway swarm and ranged from ~5% to locally ~79% in shorter transects across other swarms, which corresponds to a minimum of ~12 km of extension. The dominant NE-striking dikes are compatible with the dextral motion on the N- to NW-striking (~355-320°) regional strike-slip faults. Some of the W-NW- to NW-striking dikes were arguably influenced by pre-existing faults, shear fractures, and foliations, and potentially in one swarm where both NE-and lesser W-NW-striking dikes are present, by a switch in principal stress axes induced by dike emplacement. Alternatively, the W-NW- to NW-striking dikes may reflect a younger regional strain field, as ca. 49.3-47.5 Ma U-Pb zircon ages of the NE-striking dikes are older than those of the few dated W-NW- to NW-trending dikes. In one scenario, NE-striking dikes intruded during an interval when strain mainly reflected dextral strike-slip faulting, and the younger dikes record a switch to more arc-normal extension. Diking ended as magmatism migrated into a N-S-trending belt west of the North Cascades core that marks the initiation of the ancestral Cascade arc

Multiple low-temperature skyrmionic states in a bulk chiral magnet

Magnetic skyrmions are topologically protected nanoscale spin textures with particle-like properties. In bulk cubic helimagnets, they appear under applied magnetic fields and condense spontaneously into a lattice in a narrow region of the phase diagram just below the magnetic ordering temperature, the so-called A-phase. Theory, however, predicts skyrmions to be locally stable in a wide range of magnetic fields and temperatures. Our neutron diffraction measurements reveal the formation of skyrmion states in large areas of the magnetic phase diagram, from the lowest temperatures up to the A-phase. We show that nascent and disappearing spiral states near critical lines catalyze topological charge changing processes, leading to the formation and destruction of skyrmionic states at low temperatures, which are thermodynamically stable or metastable depending on the orientation and strength of the magnetic field. Skyrmions are surprisingly resilient to high magnetic fields: the memory of skyrmion lattice states persists in the field polarized state, even when the skyrmion lattice signal has disappeared. These findings highlight the paramount role of magnetic anisotropies in stabilizing skyrmionic states and open up new routes for manipulating these quasi-particles towards energy-efficient spintronics applications

Intermolecular Structure Determination of Amyloid Fibrils with 2 Magic-Angle Spinning and Dynamic Nuclear Polarization NMR

We describe magic-angle spinning NMR experiments designed to elucidate the interstrand architecture of amyloid fibrils. Three methods are introduced for this purpose, two being based on the analysis of long-range [superscript 13]C–[superscript 13]C correlation spectra and the third based on the identification of intermolecular interactions in [superscript 13]C–[superscript 15]N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3 or PI3K-SH3), that efficient [superscript 13]C–[superscript 13]C correlation spectra display a resonance degeneracy that establishes a parallel, in-register alignment of the proteins in the amyloid fibrils. In addition, this degeneracy can be circumvented to yield direct intermolecular constraints. The [superscript 13]C–[superscript 13]C experiments are corroborated by [superscript 15]N–[superscript 13]C correlation spectra obtained from a mixed [[superscript 15]N,[superscript 12]C]/[[superscript 14]N,[superscript 13]C] sample which directly quantify interstrand distances. Furthermore, when the spectra are recorded with signal enhancement provided by dynamic nuclear polarization (DNP) at 100 K, we demonstrate a dramatic increase (from 23 to 52) in the number of intermolecular [superscript 15]N–[superscript 13]C constraints detectable in the spectra. The increase in the information content is due to the enhanced signal intensities and to the fact that dynamic processes, leading to spectral intensity losses, are quenched at low temperatures. Thus, acquisition of low temperature spectra addresses a problem that is frequently encountered in MAS spectra of proteins. In total, the experiments provide 111 intermolecular [superscript 13]C–[superscript 13]C and [superscript 15]N–[superscript 13]C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.National Institutes of Health (U.S.) (Grant EB-003151)National Institutes of Health (U.S.) (Grant EB-002804)National Institutes of Health (U.S.) (Grant EB-002026

Knowledge Translation Consensus Conference: Research Methods

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72708/1/j.1553-2712.2007.tb02378.x.pd

MARCKS phosphorylation is modulated by a peptide mimetic of MARCKS effector domain leading to increased radiation sensitivity in lung cancer cell lines

Lung cancer is the leading cause of cancer-associated mortality in the United States. Kinase hyperactivation is a known mechanism of tumorigenesis. The phosphorylation status of the plasma membrane-associated protein myristoylated alanine rich C-kinase substrate (MARCKS) effector domain (ED) was previously established as being important in the sensitivity of lung cancer to radiation. Specifically, when MARCKS ED was in a non-phosphorylated state, lung cancer cells were more susceptible to ionizing radiation and experienced prolonged double-strand DNA breaks. Additional studies demonstrated that the phosphorylation status of MARCKS ED is important for gene expression and in vivo tumor growth. The present study used a peptide mimetic of MARCKS ED as a therapeutic intervention to modulate MARCKS phosphorylation. Culturing A549, H1792 and H1975 lung cancer cell lines with the MARCKS ED peptide led to reduced levels of phosphorylated MARCKS and phosphorylated Akt serine/threonine kinase 1. Further investigation demonstrated that the peptide therapy was able to reduce lung cancer cell proliferation and increase radiation sensitivity. In addition, the MARCKS peptide therapy was able to prolong double-strand DNA breaks following ionizing radiation exposure. The results of the present study demonstrate that a peptide mimetic of MARCKS ED is able to modulate MARCKS phosphorylation, leading to an increase in sensitivity to radiation. Keywords: lung cancer, myristoylated alanine rich C-kinase substrate, radiation sensitivity, effector domain, peptide mimeti

EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells

Lamellipodial protrusion and directional migration of carcinoma cells towards chemoattractants, such as epidermal growth factor (EGF), depend upon the spatial and temporal regulation of actin cytoskeleton by actin-binding proteins (ABPs). It is generally hypothesized that the activity of many ABPs are temporally and spatially regulated by PIP2; however, this is mainly based on in vitro–binding and structural studies, and generally in vivo evidence is lacking. Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP2 in living cells. We show that EGF induces a rapid loss of PIP2 through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin. Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation. Moreover, our data provide evidence for how PLC is involved in the formation of protrusions in breast carcinoma cells during chemotaxis and metastasis towards EGF

The Borrelia afzelii outer membrane protein BAPKO_0422 binds human Factor-H and is predicted to form a membrane-spanning beta-barrel

The deep evolutionary history of the Spirochetes places their branch point early in the evolution of the diderms, before the divergence of the present day Proteobacteria. As a Spirochete, the morphology of the Borrelia cell envelope shares characteristics of both Gram-positive and Gram-negative bacteria. A thin layer of peptidoglycan, tightly associated with the cytoplasmic membrane is surrounded by a more labile outer membrane (OM). This OM is rich in lipoproteins but with few known integral membrane proteins. The OmpA domain is an eight-stranded membrane-spanning β-barrel, highly conserved among the Proteobacteria but so far unknown in the Spirochetes. In the present work we describe the identification of four novel OmpA-like β-barrels from Borrelia afzelii, the most common cause of erythema migrans rash in Europe. Structural characterisation of one these proteins (BAPKO_0422) by small angle X-ray scattering (SAXS) and circular dichroism indicate a compact globular structure rich in β-strand consistent with a monomeric β-barrel. Ab initio molecular envelopes calculated from the scattering profile are consistent with homology models and demonstrate that BAPKO_0422 adopts a peanut shape with dimensions 25 x 45 Å. Deviations from the standard C-terminal signature sequence are apparent; in particular the C-terminal Phe residue commonly found in Proteobacterial OM proteins is replaced by Ile/Leu or Asn. BAPKO_0422 is demonstrated to bind human factor-H and therefore may contribute to immune evasion by inhibition of the complement response. Encoded by chromosomal genes, these proteins are highly conserved between Borrelia subspecies and may be of diagnostic or therapeutic value