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Messenger RNA (mRNA) therapies have recently gained tremendous traction with the approval of mRNA vaccines for the prevention of SARS-CoV-2 infection. However, manufacturing challenges have complicated large scale mRNA production, which is necessary for the clinical viability of these therapies. Not only can the incorporation of the required 5' 7-methylguanosine cap analog be inefficient and costly, in vitro transcription (IVT) using wild-type T7 RNA polymerase generates undesirable double-stranded RNA (dsRNA) byproducts that elicit adverse host immune responses and are difficult to remove at large scale. To overcome these challenges, we have engineered a novel RNA polymerase, T7-68, that co-transcriptionally incorporates both di- and tri-nucleotide cap analogs with high efficiency, even at reduced cap analog concentrations. We also demonstrate that IVT products generated with T7-68 have reduced dsRNA content.

Objective: The Alertgy noninvasive continuous glucose monitor (ANICGM) is a novel wristband device that reports glucose levels without entailing skin puncture. This study evaluated the performance of the ANICGM compared to a Food and Drug Administration-approved glucose meter in patients with type 2 diabetes., Methods: The ANICGM device measures changes in the electromagnetic field generated by its sensor to produce a dielectric spectrum. The data contained within this spectrum are used in tandem with machine learning algorithms to estimate blood glucose levels. Values from the ANICGM were collected, sent to the Alertgy lab, formatted, and compared with fingerstick blood glucose levels, which were measured using the Accuchek Inform II glucometer. Fifteen patients completed three 120-minute sessions. The mean absolute relative difference (MARD) was calculated., Results: MARD values were compared between study days 2 and 3. The MARD for day 2 was 18.5% (95% CI, 12.8-42.2%), and the MARD for day 3 was 15.3% (95% CI, 12.3-18.4%). The difference in the MARD between days 2 and 3 was not statistically significant (P = .210)., Conclusion: The resulting MARDs suggest that further investigation into the use of dielectric spectroscopy for glucose monitoring should be explored., (Copyright © 2021 AACE. Published by Elsevier Inc. All rights reserved.)

Health communications may not reach intended populations due to cultural and language barriers. These barriers may prohibit consumers from understanding information needed to make informed health decisions. It is important to ensure everyone-especially racial and ethnic minorities and under-served and under-represented populations-has access to information on medical products. One strategy to address this issue is to develop trainings and resources to better understand how cultural competency affects the ability to communicate effectively with racial/ethnic minorities. The FDA's Office of Minority Health & Health Equity developed a 3-module training to (1) increase staff knowledge of the role that cultural competency plays in determining health communication messages and channels and (2) provide tools to assist them in creating culturally-competent strategies and action plans. Offered on 4 occasions, the 4.5-h interactive training, grounded in adult learning and project-based learning theories, and used curricula, case studies, and multimedia to guide the discussion and group work. Participants also completed an action plan to guide their current work. Cultural competency knowledge was assessed pre- and post-training and training satisfaction was assessed post-training. Among the 53 individuals who completed the training, average knowledge increased by 13.6%. The training was a success based on anecdotal and evaluation feedback. The majority of participants indicated that they would refer their colleagues to the training and apply what they learned in their work. Participants felt the training was meaningful, applicable to their work, and provided an opportunity to learn and engage with their peers. Becoming culturally competent is a process that should be supported through ongoing training to help build a strong communications and health educator workforce with expertise in developing culturally competent messages to meet their constituents' needs.

Dgcr8 knockout embryonic stem (ES) cells lack microprocessor activity and hence all canonical microRNAs (miRNAs). These cells proliferate slowly and accumulate in G1 phase of the cell cycle. Here, by screening a comprehensive library of individual miRNAs in the background of the Dgcr8 knockout ES cells, we report that multiple ES cell-specific miRNAs, members of the miR-290 family, rescue the ES cell proliferation defect. Furthermore, rescued cells no longer accumulate in the G1 phase of the cell cycle. These miRNAs function by suppressing several key regulators of the G1-S transition. These results show that post-transcriptional regulation by miRNAs promotes the G1-S transition of the ES cell cycle, enabling rapid proliferation of these cells. Our screening strategy provides an alternative and powerful approach for uncovering the role of individual miRNAs in biological processes, as it overcomes the common problem of redundancy and saturation in the miRNA system.

Pairing between the hexamer seed region of a small interfering RNA (siRNA) guide strand (nucleotides 2-7) and complementary sequences in the 3' UTR of mature transcripts has been implicated as an important element in off-target gene regulation and false positive phenotypes. To better understand the association between seed sequences and off-target profiles we performed an analysis of all possible (4096) hexamers and identified a nonuniform distribution of hexamer frequencies across the 3' UTR transcriptome. Subsequent microarray analysis of cells transfected with siRNAs having seeds with low, medium, or high seed complement frequencies (SCFs) revealed that duplexes with low SCFs generally induced fewer off-targets and off-target phenotypes than molecules with more abundant 3' UTR complements. These findings provide the first experimentally validated strategy for designing siRNAs with enhanced specificity and allow for more accurate interpretation of high throughput screening data generated with existing siRNA/shRNA collections.

Since their discovery as key regulators of early animal development, microRNAs now are recognized as widespread regulators of gene expression. Despite their abundance, little is known regarding the regulation of microRNA biogenesis. We show that three highly conserved muscle-specific microRNAs, miR-1, miR-133 and miR-206, are robustly induced during the myoblast-myotube transition, both in primary human myoblasts and in the mouse mesenchymal C2C12 stem cell line. These microRNAs were not induced during osteogenic conversion of C2C12 cells. Moreover, both loci encoding miR-1, miR-1-1, and miR-1-2, and two of the three encoding miR-133, miR-133a-1 and miR-133a-2, are strongly induced during myogenesis. Some of the induced microRNAs are in intergenic regions, whereas two are transcribed in the opposite direction to the nonmuscle-specific gene in which they are embedded. By using CHIP analysis, we demonstrate that the myogenic factors Myogenin and MyoD bind to regions upstream of these microRNAs and, therefore, are likely to regulate their expression. Because miR-1 and miR-206 are predicted to repress similar mRNA targets, our work suggests that induction of these microRNAs is important in regulating the expression of muscle-specific proteins.

Off-target gene silencing can present a notable challenge in the interpretation of data from large-scale RNA interference (RNAi) screens. We performed a detailed analysis of off-targeted genes identified by expression profiling of human cells transfected with small interfering RNA (siRNA). Contrary to common assumption, analysis of the subsequent off-target gene database showed that overall identity makes little or no contribution to determining whether the expression of a particular gene will be affected by a given siRNA, except for near-perfect matches. Instead, off-targeting is associated with the presence of one or more perfect 3' untranslated region (UTR) matches with the hexamer or heptamer seed region (positions 2-7 or 2-8) of the antisense strand of the siRNA. These findings have strong implications for future siRNA design and the application of RNAi in high-throughput screening and therapeutic development.

MicroRNAs (miRNAs) are an abundant class of gene regulatory molecules (reviewed in refs 1, 2). Although computational work indicates that miRNAs repress more than a third of human genes, their roles in vertebrate development are only now beginning to be determined. Here we show that miR-196 acts upstream of Hoxb8 and Sonic hedgehog (Shh) in vivo in the context of limb development, thereby identifying a previously observed but uncharacterized inhibitory activity that operates specifically in the hindlimb. Our data indicate that miR-196 functions in a fail-safe mechanism to assure the fidelity of expression domains that are primarily regulated at the transcriptional level, supporting the idea that many vertebrate miRNAs may function as a secondary level of gene regulation.

MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally. To block all miRNA formation in zebrafish, we generated maternal-zygotic dicer (MZdicer) mutants that disrupt the Dicer ribonuclease III and double-stranded RNA-binding domains. Mutant embryos do not process precursor miRNAs into mature miRNAs, but injection of preprocessed miRNAs restores gene silencing, indicating that the disrupted domains are dispensable for later steps in silencing. MZdicer mutants undergo axis formation and differentiate multiple cell types but display abnormal morphogenesis during gastrulation, brain formation, somitogenesis, and heart development. Injection of miR-430 miRNAs rescues the brain defects in MZdicer mutants, revealing essential roles for miRNAs during morphogenesis.

MicroRNAs (miRNAs) are short endogenous RNAs known to post-transcriptionally repress gene expression in animals and plants. A microarray profiling survey revealed the expression patterns of 175 human miRNAs across 24 different human organs. Our results show that proximal pairs of miRNAs are generally coexpressed. In addition, an abrupt transition in the correlation between pairs of expressed miRNAs occurs at a distance of 50 kb, implying that miRNAs separated by <50 kb typically derive from a common transcript. Some microRNAs are within the introns of host genes. Intronic miRNAs are usually coordinately expressed with their host gene mRNA, implying that they also generally derive from a common transcript, and that in situ analyses of host gene expression can be used to probe the spatial and temporal localization of intronic miRNAs.

Background: Early identification of acute MI and prompt intervention can improve clinical outcomes. It would be valuable to identify a method that could allow the earliest possible detection of myocardial injury or ischemia., Methods and Results: This article reports one of the first clinical investigations to examine the ability of an intracardiac right ventricular (RV) electrode to identify the early onset of myocardial ischemia/injury in a cohort of patients undergoing balloon occlusion of a coronary artery during percutaneous transluminal coronary angioplasty. The primary data set for analysis included observations from 14 patients with 17 lesions, with a matched comparison of a V6 surface lead and the RV to left upper chest, "intracardiac" lead. The intracardiac lead was sensitive in detecting myocardial injury current/ischemia. There was a 36.4+/-5.6% ST-segment shift, relative to the amplitude of the QRS complex, in the intracardiac lead at 2 min, compared with a 10.1+/-1.9% ST shift from a surface lead (P=.00011). The RV to left upper chest lead detected a >10% shift in ST segment within 2 min in 17 (100%) of 17 cases vs. 8 (47%) of 17 for a V6 surface lead. The intracardiac lead provided detection of ischemia in all three major epicardial coronary distributions., Conclusions: This study demonstrates the ability of an intracardiac (RV apex to left upper chest) lead to rapidly detect myocardial ischemia/injury during acute coronary occlusion in the setting of balloon angioplasty. The results of this study suggest that a simple implantable system resembling a ventricular pacemaker could be programmed to assist in the very early diagnosis of acute myocardial infarction.

Purpose: This study was designed to compare the rates of subacute stent thrombosis (SAT) among patients receiving heparin-coated stents to patients receiving bare-metal stents in real world, contemporary coronary interventions., Background: Controlled trials with heparin-coated coronary stents have shown a trend toward decreased rates of SAT., Methods and Results: The data in this study were collected from a single, large cardiac center over a period of 9 months. All patients who underwent coronary stent implantation during this 9-month period were included in the study (1,288 patients; 1,366 procedures; 2,231 stents). All patients were treated with aspirin and clopidogrel (or ticlopidine) after stenting. Primary thrombotic outcome was defined as angiographically documented SAT and/or sudden unexplained cardiac death (SCD) within 30 days of the procedure. Follow-up data (1,264/1,276 patients) were obtained in 99% of patients. A total of 337 patients received 543 heparin-coated stents (BX Velocity Hepacoat) and 939 patients received bare-metal stents (1,688 stents). SAT was seen in 25/1,024 procedures (2.44%) in the bare-metal stent group and 1/342 procedures (0.29%) in the heparin-coated stent group. Primary thrombotic outcomes (SAT or SCD) were observed in 31/1,024 procedures (3.03%) in the bare-metal stent cohort and in 2/342 procedures (0.58%) in the heparin-coated stent group. The vast majority (96%) of the patients who had SAT within 30 days had initial stent placement for an acute coronary syndrome (p<0.0001)., Conclusion: This large, single-center registry demonstrates a significant reduction of SAT using heparin-coated stents compared to bare-metal stents in real world coronary interventions.

Objectives: A discrepancy exists in the medical literature as to what effect intravascular ultrasound (IVUS)-guided stent deployment has on target vessel revascularization (TVR) at 6 months. The major endpoints of this study are the need for TVR, defined as clinically driven repeat interventional or surgical therapy of the index vessel at 6 months and major adverse cardiac events., Methods: One hundred interventional stent cases (50 IVUS-guided, 50 non-IVUS guided) were randomly selected in a 6-month period (January to June 2001) for review by measurement of minimal luminal diameter (MLD) pre- and post-intervention. Seventy males and 30 females were distributed among the 2 groups. There were a total of 135 lesions (70 IVUS-guided, 65 non-IVUS guided) in the 2 groups. A 6-month follow-up chart review was performed following the initial stenting., Results: At 6-month follow-up, there were 2 deaths in the IVUS-guided group and 3 deaths in the non-IVUS guided group (p=NS). All deaths were cardiovascular in nature. Post-procedure MLD was 3.58+/-0.08 mm for the IVUS-guided group and 2.88+/-0.09 mm for the non-IVUS guided group [t=5.7 (df, 133); p<0.001]. Ten of 70 IVUS-guided lesions (14.3%) and 3 of 65 non-IVUS guided lesions (4.2%) underwent TVR within the 6-month study period (Chi square=3.62; p=0.057)., Conclusion: In this population, IVUS-guided stent deployment does not appear to reduce either the need for TVR or overall cardiovascular mortality at 6 months. The added expense of IVUS does not appear to be warranted.

We have used a combination of in vitro selection and rational design to generate ribozymes that form a stable phosphoamide bond between the 5' terminus of an RNA and a specific polypeptide. This reaction differs from that of previously identified ribozymes, although the product is analogous to the enzyme-nucleotidyl intermediates isolated during the reactions of certain proteinaceous enzymes, such as guanyltransferase, DNA ligase, and RNA ligase. Comparative sequence analysis of the isolated ribozymes revealed that they share a compact secondary structure containing six stems arranged in a four-helix junction and branched pseudoknot. An optimized version of the ribozyme reacts with substrate-fusion proteins, allowing it to be used to attach RNA tags to proteins both in vitro and within bacterial cells, suggesting a simple way to tag a specific protein with amplifiable information.

Despite improvements in catheter-based revascularization outcomes, coronary interventionalists face difficult challenges in the treatment of the thrombus-laden coronary lesion. In this report, we describe the use of the Acolysis device, which utilizes high-frequency (41.9 kHz) ultrasonic energy to vibrate a small metal tip at the end of a 4.5 Fr catheter to treat two thrombotically occluded saphenous vein grafts in two patients. In both cases, the Acolysis device provided normalization of flow with angiographically evident dissolution of thrombus and excellent acute angiographic and clinical results. We conclude that in these two selected cases the Acolysis device was used safely and effectively for thrombus debulking as an adjunct to stenting in diseased saphenous vein bypass grafts.

Background: The Rex protein of the human T cell leukemia virus type 1 (HTLV-1) belongs to a family of proteins that use arginine-rich motifs (ARMs) to recognize their RNA targets. Previously, an in vitro selected RNA aptamer sequence was identified that mediates mRNA transport in vivo when placed in the primary binding site on stem-loop IID of the Rex response element. We present the solution structure of the HTLV-1 arginine-rich Rex peptide bound to its RNA aptamer target determined by multidimensional heteronuclear NMR spectroscopy., Results: The Rex peptide in a predominantly extended conformation threads through a channel formed by the shallow and widened RNA major groove and a looped out guanine. The RNA aptamer contains three stems separated by a pair of two-base bulges, and adopts an unanticipated fold in which both junctional sites are anchored through base triple formation. Binding specificity is associated with intermolecular hydrogen bonding between guanidinium groups of three non-adjacent arginines and the guanine base edges of three adjacent G.C pairs., Conclusions: The extended S-shaped conformation of the Rex peptide, together with previous demonstrations of a beta-hairpin conformation for the bovine immunodeficiency virus (BIV) Tat peptide and an alpha-helical conformation for the human immunodeficiency virus (HIV) Rev peptide in complex with their respective RNA targets, expands our understanding of the strategies employed by ARMs for adaptive recognition and highlights the importance of RNA tertiary structure in accommodating minimalist elements of protein secondary structure.

RNA molecules that bind tightly and specifically to a Rex fusion protein have been isolated from a conformationally constrained pool of random sequence RNAs. The anti-Rex aptamers effectively mimic several features of the wild-type Rex-binding element (XBE). The highest-affinity aptamers effectively compete with the wild-type XBE for binding to the RNA-binding domain of Rex, an arginine-rich motif (ARM), but do not bind to the functionally analogous Rev protein or its ARM. However, characteristic sequence and structural motifs found in some of the anti-Rex aptamers may provide insights into how the Rex protein can interact with other viral RNAs, such as the Rev-responsive element. The anti-Rex aptamers can functionally substitute for the XBE in vivo, a result which supports a previously proposed model for mRNA transport in which the viral genome serves as a platform for assembling a nucleoprotein complex that can co-opt the cellular transport apparatus. Overall, these studies suggest that anti-Rex aptamers may serve as RNA decoys of the Rex protein.

Interactions between Rev and the Rev-responsive element (RRE) control the order, rate, and extent of gene expression in human immunodeficiency virus type 1. Rev decoys may therefore prove to be useful RNA therapeutics for the treatment of AIDS. To improve upon the current generation of Rev decoys that bind single Rev molecules, it would be useful to generate polyvalent Rev decoys that could bind multiple Rev molecules. J. Kjems and P. A. Sharp (J. Virol. 67:4769-4776, 1993) originally constructed functional polyvalent Rev decoys, but the structural context of these polyvalent decoys remains unclear, and it has been argued that the individual decoys were either structurally discrete (Kjems and Sharp, J. Virol. 67:4769-4776, 1993) or were part of an extended helix (R. W. Zemmel et al., Mol. Biol. 258:763-777, 1996). To resolve the differences between these models, we have designed and synthesized concatemers of Rev-binding elements (RBEs) that fold to form multiple, discrete, high-affinity Rev-binding sites. We find that the concatenated RBEs can facilitate the cytoplasmic transport of viral mRNAs and therefore likely bind multiple Rev molecules. These artificial RREs may simultaneously sequester Rev and hinder access to the cellular transport machinery.

In vitro selection, or SELEX, has been used both to characterize the interaction of natural nucleic acids with proteins and to generate novel nucleic acid-binding species, or aptamers. Although numerous reports have demonstrated the power of the technique, they have not expanded on the methodologies that can be used for selection. This review focuses on the considerations and problems involved in selecting protein-binding aptamers from a random-sequence RNA pool. As an illustration, we describe two approaches to selecting aptamers to a particular target, the HTLV-I Rex protein. In the first, complete randomization is used to find an artificial, high-affinity RNA binding site. In the second, the contributions of individual nucleotides and/or base pairs to the natural Rex-binding element are determined by mutating the wild-type sequence and selecting active binding variants.

The Tat and Rev proteins of HIV-1 and the Rex protein of HTLV-I do not interact with their cognate ligands via a particular structural motif but instead specifically recognize RNA molecules by using agglomerations of arginine residues (1). These proteins are members of the so-called arginine-rich motif (ARM) family. There is little data to support (or contradict) the hypothesis that a few simple arginine:RNA interactions govern how ARMs recognize their viral targets. Not only is it unclear how ARM proteins other than Tat interact with their cognate RNA ligands, for the most part it is not even known how structurally complex these RNA ligands are. In order to fully explore the range of RNA sequences and structures that can bind to ARMs we have carried out in vitro genetic selections with two disparate viral proteins: Rev and Rex.