Single mRNAs visualized by ultrastructural in situ hybridization are principally localized at actin filament intersections in fibroblasts

Considerable evidence indicates that mRNA associates with structural filaments in the cell (cytoskeleton). This relationship would be an important mechanism to effect mRNA sorting since specific mRNAs could be sequestered at sites within the cell. In addition, it can provide a mechanism for spatial regulation of mRNA expression. However, the precise structural interactions between mRNA and the cytoskeleton have yet to be defined. An objective of this work was to visualize individual poly(A) mRNA molecules in situ by electron microscopy to identify their relationship to individual filaments. Poly(A) RNA and filaments were identified simultaneously using antibodies to detect hybridized probe and filaments or actin-binding proteins. In human fibroblasts, most of the poly(A) mRNA (72%) was localized within 5 nm of orthogonal networks of F-actin filaments. Poly(A) mRNA also colocalized with vimentin filaments (29%) and microtubules (\u3c 10%). The sites of mRNA localization were predominantly at filament intersections. The majority of poly(A) mRNA and polysomes colocalized with the actin crosslinking proteins, filamin, and alpha-actinin, and the elongation factor, EF-1 alpha (actin-binding protein; ABP-50). Evidence that intersections contained single mRNA molecules was provided by using a labeled oligo dT probe to prime the synthesis of cDNA in situ using reverse transcriptase. Both the poly(A) and cis sequences of the same mRNA molecule could then be visualized independently. We propose that the cytoskeletal intersection is a mRNA receptor and serves as a microdomain where mRNA is attached and functionally expressed

Vesicle shape, molecular tilt, and the suppression of necks

Can the presence of molecular-tilt order significantly affect the shapes of lipid bilayer membranes, particularly membrane shapes with narrow necks? Motivated by the propensity for tilt order and the common occurrence of narrow necks in the intermediate stages of biological processes such as endocytosis and vesicle trafficking, we examine how tilt order inhibits the formation of necks in the equilibrium shapes of vesicles. For vesicles with a spherical topology, point defects in the molecular order with a total strength of $+2$ are required. We study axisymmetric shapes and suppose that there is a unit-strength defect at each pole of the vesicle. The model is further simplified by the assumption of tilt isotropy: invariance of the energy with respect to rotations of the molecules about the local membrane normal. This isotropy condition leads to a minimal coupling of tilt order and curvature, giving a high energetic cost to regions with Gaussian curvature and tilt order. Minimizing the elastic free energy with constraints of fixed area and fixed enclosed volume determines the allowed shapes. Using numerical calculations, we find several branches of solutions and identify them with the branches previously known for fluid membranes. We find that tilt order changes the relative energy of the branches, suppressing thin necks by making them costly, leading to elongated prolate vesicles as a generic family of tilt-ordered membrane shapes.Comment: 10 pages, 7 figures, submitted to Phy. Rew.

Klotho pathways, myelination disorders, neurodegenerative diseases, and epigenetic drugs

In this review we outline a rationale for identifying neuroprotectants aimed at inducing endogenous Klotho activity and expression, which is epigenetic action, by definition. Such an approach should promote remyelination and/or stimulate myelin repair by acting on mitochondrial function, thereby heralding a life-saving path forward for patients suffering from neuroinflammatory diseases. Disorders of myelin in the nervous system damage the transmission of signals, resulting in loss of vision, motion, sensation, and other functions depending on the affected nerves, currently with no effective treatment. Klotho genes and their single-pass transmembrane Klotho proteins are powerful governors of the threads of life and death, true to the origin of their name, Fates, in Greek mythology. Among its many important functions, Klotho is an obligatory co-receptor that binds, activates, and/or potentiates critical fibroblast growth factor activity. Since the discovery of Klotho a little over two decades ago, it has become ever more apparent that when Klotho pathways go awry, oxidative stress and mitochondrial dysfunction take over, and age-related chronic disorders are likely to follow. The physiological consequences can be wide ranging, potentially wreaking havoc on the brain, eye, kidney, muscle, and more. Central nervous system disorders, neurodegenerative in nature, and especially those affecting the myelin sheath, represent worthy targets for advancing therapies that act upon Klotho pathways. Current drugs for these diseases, even therapeutics that are disease modifying rather than treating only the symptoms, leave much room for improvement. It is thus no wonder that this topic has caught the attention of biomedical researchers around the world.https://www.liebertpub.com/doi/10.1089/biores.2020.0004Published versio

Novel Amphiphilic Cyclobutene and Cyclobutane \u3ci\u3ecis\u3c/i\u3e-C18 Fatty Acid Derivatives Inhibit \u3ci\u3eMycobacterium avium\u3c/i\u3e subsp. \u3ci\u3eparatuberculosis\u3c/i\u3e Growth

Mycobacterium avium subspecies paratuberculosis (Map) is the etiologic agent of Johne’s disease in ruminants and has been associated with Crohn’s disease in humans. An effective control of Map by either vaccines or chemoprophylaxis is a paramount need for veterinary and possibly human medicine. Given the importance of fatty acids in the biosynthesis of mycolic acids and the mycobacterial cell wall, we tested novel amphiphilic C10 and C18 cyclobutene and cyclobutane fatty acid derivatives for Map inhibition. Microdilution minimal inhibitory concentrations (MIC) with 5 or 7 week endpoints were measured in Middlebrook 7H9 base broth media. We compared the Map MIC results with those obtained previously with Mycobacterium tuberculosis and Mycobacterium smegmatis. Several of the C18 compounds showed moderate effcacy (MICs 392 to 824 μM) against Map, while a higher level of inhibition (MICs 6 to 82 μM) was observed for M. tuberculosis for select analogs from both the C10 and C18 groups. For most of these analogs tested in M. smegmatis, their effcacy decreased in the presence of bovine or human serum albumin. Compound 5 (OA-CB, 1-(octanoic acid-8-yl)-2-octylcyclobutene) was identified as the best chemical lead against Map, which suggests derivatives with better pharmacodynamics may be of interest for evaluation in animal models

Unique opportunities for NMR methods in structural genomics

This Perspective, arising from a workshop held in July 2008 in Buffalo NY, provides an overview of the role NMR has played in the United States Protein Structure Initiative (PSI), and a vision of how NMR will contribute to the forthcoming PSI-Biology program. NMR has contributed in key ways to structure production by the PSI, and new methods have been developed which are impacting the broader protein NMR community

Patient-Reported Outcomes to Support Medical Product Labeling Claims: FDA Perspective

AbstractThis article concerns development and use of patient-reported outcomes (PROs) in clinical trials to evaluate medical products. A PRO is any report coming directly from patients, without interpretation by physicians or others, about how they function or feel in relation to a health condition and its therapy. PRO instruments are used to measure these patient reports. PROs provide a unique perspective on medical therapy, because some effects of a health condition and its therapy are known only to patients. Properly developed and evaluated PRO instruments also have the potential to provide more sensitive and specific measurements of the effects of medical therapies, thereby increasing the efficiency of clinical trials that attempt to measure the meaningful treatment benefits of those therapies. Poorly developed andevaluated instruments may provide misleading conclusions or data that cannot be used to support product labeling claims. We review selected major challenges from Food and Drug Administration's perspective in using PRO instruments, measures, and end points to support treatment benefit claims in product labeling. These challenges highlight the need for sponsors to formulate desired labeling claim(s) prospectively, to acquire and document information needed to support these claim(s), and to identify existing instruments or develop new and more appropriate PRO instruments for evaluating treatment benefit in the defined population in which they will seek claims

Probing complex RNA structures by mechanical force

RNA secondary structures of increasing complexity are probed combining single molecule stretching experiments and stochastic unfolding/refolding simulations. We find that force-induced unfolding pathways cannot usually be interpretated by solely invoking successive openings of native helices. Indeed, typical force-extension responses of complex RNA molecules are largely shaped by stretching-induced, long-lived intermediates including non-native helices. This is first shown for a set of generic structural motifs found in larger RNA structures, and then for Escherichia coli's 1540-base long 16S ribosomal RNA, which exhibits a surprisingly well-structured and reproducible unfolding pathway under mechanical stretching. Using out-of-equilibrium stochastic simulations, we demonstrate that these experimental results reflect the slow relaxation of RNA structural rearrangements. Hence, micromanipulations of single RNA molecules probe both their native structures and long-lived intermediates, so-called "kinetic traps", thereby capturing -at the single molecular level- the hallmark of RNA folding/unfolding dynamics.Comment: 9 pages, 9 figure

The Role of Citizens in Detecting and Responding to a Rapid Marine Invasion

Documenting and responding to species invasions requires innovative strategies that account for ecological and societal complexities. We used the recent expansion of Indo-Pacific lionfish (Pterois volitans/miles) throughout northern Gulf of Mexico coastal waters to evaluate the role of stakeholders in documenting and responding to a rapid marine invasion. We coupled an online survey of spearfishers and citizen science monitoring programs with traditional fishery-independent data sources and found that citizen observations documented lionfish 1–2 years earlier and more frequently than traditional reef fish monitoring programs. Citizen observations first documented lionfish in 2010 followed by rapid expansion and proliferation in 2011 (+367%). From the survey of spearfishers, we determined that diving experience and personal observations of lionfish strongly influenced perceived impacts, and these perceptions were powerful predictors of support for initiatives. Our study demonstrates the value of engaging citizens for assessing and responding to large-scale and time-sensitive conservation problems