A General RNA Motif for Cellular Transfection

We have developed a selection scheme to generate nucleic acid sequences that recognize and directly internalize into mammalian cells without the aid of conventional delivery methods. To demonstrate the generality of the technology, two independent selections with different starting pools were performed against distinct target cells. Each selection yielded a single highly functional sequence, both of which folded into a common core structure. This internalization signal can be adapted for use as a general purpose reagent for transfection into a wide variety of cell types including primary cells

PANC Study (Pancreatitis: A National Cohort Study): national cohort study examining the first 30 days from presentation of acute pancreatitis in the UK

Abstract Background Acute pancreatitis is a common, yet complex, emergency surgical presentation. Multiple guidelines exist and management can vary significantly. The aim of this first UK, multicentre, prospective cohort study was to assess the variation in management of acute pancreatitis to guide resource planning and optimize treatment. Methods All patients aged greater than or equal to 18 years presenting with acute pancreatitis, as per the Atlanta criteria, from March to April 2021 were eligible for inclusion and followed up for 30 days. Anonymized data were uploaded to a secure electronic database in line with local governance approvals. Results A total of 113 hospitals contributed data on 2580 patients, with an equal sex distribution and a mean age of 57 years. The aetiology was gallstones in 50.6 per cent, with idiopathic the next most common (22.4 per cent). In addition to the 7.6 per cent with a diagnosis of chronic pancreatitis, 20.1 per cent of patients had a previous episode of acute pancreatitis. One in 20 patients were classed as having severe pancreatitis, as per the Atlanta criteria. The overall mortality rate was 2.3 per cent at 30 days, but rose to one in three in the severe group. Predictors of death included male sex, increased age, and frailty; previous acute pancreatitis and gallstones as aetiologies were protective. Smoking status and body mass index did not affect death. Conclusion Most patients presenting with acute pancreatitis have a mild, self-limiting disease. Rates of patients with idiopathic pancreatitis are high. Recurrent attacks of pancreatitis are common, but are likely to have reduced risk of death on subsequent admissions. </jats:sec

Evolutionarily Repurposed Networks Reveal the Well-Known Antifungal Drug Thiabendazole to Be a Novel Vascular Disrupting Agent

Hye Ji Cha is with UT Austin, Michelle Byrom is with UT Austin, Paul E. Mead is with St. Jude Children's Research Hospital, Andrew D. Ellington is with UT Austin, John B. Wallingford is with UT Austin, Edward M. Marcotte is with UT Austin.Studies in diverse organisms have revealed a surprising depth to the evolutionary conservation of genetic modules. For example, a systematic analysis of such conserved modules has recently shown that genes in yeast that maintain cell walls have been repurposed in vertebrates to regulate vein and artery growth. We reasoned that by analyzing this particular module, we might identify small molecules targeting the yeast pathway that also act as angiogenesis inhibitors suitable for chemotherapy. This insight led to the finding that thiabendazole, an orally available antifungal drug in clinical use for 40 years, also potently inhibits angiogenesis in animal models and in human cells. Moreover, in vivo time-lapse imaging revealed that thiabendazole reversibly disassembles newly established blood vessels, marking it as vascular disrupting agent (VDA) and thus as a potential complementary therapeutic for use in combination with current anti-angiogenic therapies. Importantly, we also show that thiabendazole slows tumor growth and decreases vascular density in preclinical fibrosarcoma xenografts. Thus, an exploration of the evolutionary repurposing of gene networks has led directly to the identification of a potential new therapeutic application for an inexpensive drug that is already approved for clinical use in humans.The authors acknowledge funding from the Texas Institute for Drug and Diagnostic Development and Cancer Prevention & Research Institute of Texas (CPRIT), as well as funding to EMM from the US National Science Foundation, US National Institutes of Health, Welch Foundation (F1515), and the Packard Foundation; to PEM from the US NIH and ALSAC; and to JBW from the NIH/NIGMS and The Burroughs Wellcome Fund. JBW is an Early Career Scientist of the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Cellular and Molecular Biolog

Cellular reagents for diagnostics and synthetic biology.

We have found that the overproduction of enzymes in bacteria followed by their lyophilization leads to 'cellular reagents' that can be directly used to carry out numerous molecular biology reactions. We demonstrate the use of cellular reagents in a variety of molecular diagnostics, such as TaqMan qPCR with no diminution in sensitivity, and in synthetic biology cornerstones such as the Gibson assembly of DNA fragments, where new plasmids can be constructed solely based on adding cellular reagents. Cellular reagents have significantly reduced complexity and cost of production, storage and implementation, features that should facilitate accessibility and use in resource-poor conditions

Engineering Signaling Aptamers That Rely on Kinetic Rather Than Equilibrium Competition

During the past decade, aptasensors have largely been designed on the basis of the notion that ligand-modulated equilibration between aptamer conformations could be exploited for sensing. One implementation of this strategy has been to denature the aptamer with an antisense oligonucleotide, wait for dissociation of the antisense oligonucleotide, and stabilize the folded, signaling conformer with a ligand. However, there is a large kinetic barrier associated with releasing the oligonucleotide from the aptamer to again obtain an active, binding conformation. If the length of the antisense oligonucleotide is decreased to make dissociation from the aptamer more favorable, higher background signals are observed. To improve the general methodology for developing aptasensors, we have developed a novel and robust strategy for aptasensor design in which an oligonucleotide kinetically competes with the ligand for binding rather than having to be released from a stable duplex. While the oligonucleotide can induce conformational change, it initially chooses between the aptamer and a molecular beacon (MB), a process that does not require a lengthy pre-equilibration. Using an anti-ricin aptamer as a starting point, we developed a “competitive” aptasensor with a measured limit of detection (LOD) of 30 nM with an optical readout and as low as 3 nM for ricin toxin A-chain (RTA) detection on an electrochemical platform

TBZ significantly disrupts tube formation in cultured human umbilical vein endothelial cells (HUVECs), an in vitro capillary model.

<p>Here, we show effects of 1% DMSO-treated control (A) versus 1% DMSO, 100 µM TBZ (B) and 1% DMSO, 250 µM TBZ (C). Scale bar, 100 µm. (D) Tube disruption is dose-dependent and comparable to that from silencing known pro-angiogenic gene <i>HOXA9</i>.</p

Blood vessel density is significantly reduced within TBZ-treated tumors.

<p>(A) and (B) show tumor vasculature visualized by immunohistochemistry of microdissected tumor sections using an anti-CD31 (PECAM-1) antibody staining for vasculature in the region of highest vessel density (“hot spots”; scale bar, 100 µm), and the total area of PECAM-1 staining above a fluorescence intensity threshold (arbitrary units) is quantified in (C).</p

TBZ impedes migration of HUVECs in a wound scratch assay, but treatment with the Rho Kinase inhibitor Y27632 reverses TBZ's effects.

<p>(A) The effects of 1% DMSO-treated control versus 1% DMSO, 250 µM TBZ, and 1% DMSO, 250 µM TBZ, 10 µM Y27632. Scale bar, 200 µm. (B) quantifies the dose-dependent suppression of TBZ inhibition by Y27632. Error bars represent the mean ± 1 s.d. across 3 wells (1 of 3 trials). TBZ results in disorganization of actin stress fibers, as shown in (C) for 1% DMSO-treated control versus 1% DMSO, 250 µM TBZ-treated cells. Scale bar, 20 µm.</p

Identification of candidate angiogenesis inhibitors based upon genetic interactions with a yeast gene module.

<p>(A) Summary of the gene module (modified from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001379#pbio.1001379-McGary1" target="_blank">[1]</a>). Tests of genes associated with the yeast phenotype (lovastatin sensitivity) correctly identified novel angiogenesis genes, as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001379#pbio.1001379-McGary1" target="_blank">[1]</a> and additionally shown in (B) for the gene <i>rab11b</i>. Morpholino (MO) knockdown of <i>rab11B</i> induces vascular defects in developing <i>Xenopus laevis</i> (frog) embryos, measured by in situ hybridization versus marker gene <i>erg</i>. ISV, intersomitic vein; PCV, posterior cardinal vein; VV, vitellin vein. (C) In an unbiased hierarchical clustering of compounds by their synthetic genetic interaction profiles with yeast genes (analyzing data from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001379#pbio.1001379-Hillenmeyer1" target="_blank">[13]</a>), the action of TBZ is among those interacting with this gene module and also most similar to lovastatin, the signature compound affiliated with the angiogenesis gene module; hence, TBZ is a likely candidate angiogenesis inhibitor. Here, complete linkage clustering employing uncentered correlation coefficients is shown; additional clustering methods are illustrated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001379#pbio.1001379.s002" target="_blank">Figure S2</a>.</p

TBZ slows the growth of human HT1080 fibrosarcoma xenograft tumors in athymic Cre nu/nu mice.

<p>Tumors are significantly reduced in size in TBZ-treated animals (A), shown in (B) biopsied from mice after 27 d of 50 mg/kg (corresponding to 250 µM) TBZ treatment, and quantified in (C) and (D) (1 of 2 trials).</p