De novo gene synthesis by an antiviral reverse transcriptase.

Bacteria defend themselves from viral infection using diverse immune systems, many of which sense and target foreign nucleic acids. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this immune strategy by instead leveraging DNA synthesis, but the identities and functions of their DNA products remain largely unknown. Here we show that DRT2 systems execute an unprecedented immunity mechanism that involves de novo gene synthesis via rolling-circle reverse transcription of a non-coding RNA (ncRNA). Unbiased profiling of RT-associated RNA and DNA ligands in DRT2-expressing cells revealed that reverse transcription generates concatenated cDNA repeats through programmed template jumping on the ncRNA. The presence of phage then triggers second-strand cDNA synthesis, leading to the production of long double-stranded DNA. Remarkably, this DNA product is efficiently transcribed, generating messenger RNAs that encode a stop codon-less, never-ending ORF (neo) whose translation causes potent growth arrest. Phylogenetic analyses and screening of diverse DRT2 homologs further revealed broad conservation of rolling-circle reverse transcription and Neo protein function. Our work highlights an elegant expansion of genome coding potential through RNA-templated gene creation, and challenges conventional paradigms of genetic information encoded along the one-dimensional axis of genomic DNA.

Bioprosthetic versus mechanical valves for mitral valve replacement in patients < 70 years: an updated pairwise meta-analysis.

BACKGROUND: The ideal conduit for mitral valve replacement (MVR) remains elusive, particularly among younger patients due to increased life expectancy. We perform a pairwise meta-analysis comparing the use of bioprosthetic valves (BPV) and mechanical mitral valves (MMV) in patients < 70 years old undergoing MVR. METHODS: We comprehensively searched medical databases to identify studies comparing the use of BPV and MMV in patients < 70 years old undergoing MVR. Pairwise meta-analysis was performed using the Mantel-Haenszel method in R version 4.0.2. Outcomes were pooled using the random effect model as risk ratios (RR) with their 95% confidence intervals (95% CI). RESULTS: 16,879 patients from 15 studies were pooled. Compared to MMV, BPV was associated with significantly higher rates of 30-day mortality (RR 1.53, p = 0.0006) but no difference in 30-day stroke (RR 0.70, p = 0.43). At a weighted mean follow-up duration of 14.1 years, BPV was associated with higher rates of long-term mortality (RR 1.28, p = 0.0054). No difference was seen between the two groups for risk of long-term stroke (RR 0.92, p = 0.67), reoperation(RR 1.72, p = 0.12), or major-bleeding (RR 0.57, p = 0.10) at a weighted mean follow-up duration of 11.7, 11.3, and 11.9 years, respectively. CONCLUSION: The use of MMV in patients < 70 undergoing MVR is associated with lower rates of 30-day/long-term mortality compared to BPV. No significant differences were observed for risk of 30-day/long-term stroke, long-term reoperation, and long-term major bleeding. These findings support the use of MMV in younger patients, although prospective, randomized trials are still needed.

Mechanism of target site selection by type V-K CRISPR-associated transposases.

CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to catalyze RNA-guided transposition of large genetic payloads. Type V-K CASTs offer potential technology advantages but lack accuracy, and the molecular basis for this drawback has remained elusive. Here, we reveal that type V-K CASTs maintain an RNA-independent, "untargeted" transposition pathway alongside RNA-dependent integration, driven by the local availability of TnsC filaments. Using cryo-electron microscopy, single-molecule experiments, and high-throughput sequencing, we found that a minimal, CRISPR-less transpososome preferentially directs untargeted integration at AT-rich sites, with additional local specificity imparted by TnsB. By exploiting this knowledge, we suppressed untargeted transposition and increased type V-K CAST specificity up to 98.1% in cells without compromising on-target integration efficiency. These findings will inform further engineering of CAST systems for accurate, kilobase-scale genome engineering applications.

A Case Series of Potential Pediatric Cyanotoxin Exposures Associated with Harmful Algal Blooms in Northwest Ohio.

Cyanobacterial harmful algal blooms (CyanoHABs) are increasing in prevalence and severity in the Great Lakes region, as well as both globally and locally. CyanoHABs have the potential to cause adverse effects on human health due to the production of cyanotoxins from cyanobacteria. Common routes of exposure include recreational exposure (swimming, skiing, and boating), ingestion, and aerosolization of contaminated water sources. Cyanotoxins have been shown to adversely affect several major organ systems contributing to hepatotoxicity, gastrointestinal distress, and pulmonary inflammation. We present three pediatric case reports that coincided with CyanoHABs exposure with a focus on presentation of illness, diagnostic work-up, and treatment of CyanoHAB-related illnesses. Potential cyanotoxin exposure occurred while swimming in the Maumee River and Maumee Bay of Lake Erie in Ohio during the summer months with confirmed CyanoHAB activity. Primary symptoms included generalized macular rash, fever, vomiting, diarrhea, and severe respiratory distress. Significant labs included leukocytosis and elevated C-reactive protein. All patients ultimately recovered with supportive care. Symptoms following potential cyanotoxin exposure coincide with multiple disease states representing an urgent need to develop specific diagnostic tests of exposure.

Minimally Invasive Versus Transcatheter Aortic Valve Replacement: An Updated Meta-Analysis and Systematic Review.

OBJECTIVE: Transcatheter aortic valve replacement (TAVR) has arisen as a viable alternative to surgery. Similarly, minimally invasive surgical aortic valve replacement (mini-SAVR), such as ministernotomy and minithoracotomy, have also gained interest. We conducted a pairwise meta-analysis to further investigate the efficacy of TAVR versus mini-SAVR. METHODS: Medical databases were comprehensively searched for studies comparing TAVR with a mini-SAVR modality, defined as minimally invasive aortic surgery, ministernotomy, minithoracotomy, or rapid-deployment or sutureless SAVR. Random-effects meta-analysis was conducted using the generic inverse variance method. Primary outcomes included 30-day mortality, midterm mortality, 30-day stroke, acute kidney injury (AKI), paravalvular leak (PVL), new permanent pacemaker (PPM), new-onset atrial fibrillation, and postintervention mean and peak valve pressure gradients and were pooled as risk ratio (RR), mean difference (MD), or hazard ratio (HR) with 95% confidence interval (CI). RESULTS: A total of 5,071 patients (2,505 mini-SAVR vs 2,566 TAVR) from 12 studies were pooled. Compared with TAVR, mini-SAVR showed significantly lower rates of both 30-day (RR = 0.63, 95% CI: 0.42 to 0.96, P = 0.03) and midterm mortality at 4 years of follow-up (HR = 0.76, 95% CI: 0.67 to 0.87, P < 0.001). Furthermore, mini-SAVR was protective against 30-day PVL (RR = 0.07, 95% CI: 0.04 to 0.13, P < 0.001) and new PPM (RR = 0.25, 95% CI: 0.11 to 0.57, P < 0.001). Conversely, TAVR was protective against 30-day AKI (RR = 1.67, 95% CI: 1.20 to 2.32, P = 0.002) and postinterventional mean gradients (MD = 1.65, 95% CI: 0.25 to 3.05, P = 0.02). No difference was observed for 30-day stroke (RR = 0.84, 95% CI: 0.56 to 1.24, P = 0.38), new-onset atrial fibrillation (RR = 1.79, 95% CI: 0.93 to 3.44, P = 0.08), or postinterventional peak gradients (MD = 3.24, 95% CI: -1.10 to 7.59, P = 0.14). CONCLUSIONS: Compared with TAVR, mini-SAVR was protective against 30-day and midterm mortality, 30-day PVL, and new permanent pacemaker, while TAVR patients had lower 30-day AKI. Future randomized trials comparing the efficacy of mini-SAVR approaches with TAVR are needed.

Mechanism of target site selection by type V-K CRISPR-associated transposases.

Unlike canonical CRISPR-Cas systems that rely on RNA-guided nucleases for target cleavage, CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to facilitate RNA-guided transposition of large genetic payloads. Type V-K CASTs offer several potential upsides for genome engineering, due to their compact size, easy programmability, and unidirectional integration. However, these systems are substantially less accurate than type I-F CASTs, and the molecular basis for this difference has remained elusive. Here we reveal that type V-K CASTs undergo two distinct mobilization pathways with remarkably different specificities: RNA-dependent and RNA-independent transposition. Whereas RNA-dependent transposition relies on Cas12k for accurate target selection, RNA-independent integration events are untargeted and primarily driven by the local availability of TnsC filaments. The cryo-EM structure of the untargeted complex reveals a TnsB-TnsC-TniQ transpososome that encompasses two turns of a TnsC filament and otherwise resembles major architectural aspects of the Cas12k-containing transpososome. Using single-molecule experiments and genome-wide meta-analyses, we found that AT-rich sites are preferred substrates for untargeted transposition and that the TnsB transposase also imparts local specificity, which collectively determine the precise insertion site. Knowledge of these motifs allowed us to direct untargeted transposition events to specific hotspot regions of a plasmid. Finally, by exploiting TnsB's preference for on-target integration and modulating the availability of TnsC, we suppressed RNA-independent transposition events and increased type V-K CAST specificity up to 98.1%, without compromising the efficiency of on-target integration. Collectively, our results reveal the importance of dissecting target site selection mechanisms and highlight new opportunities to leverage CAST systems for accurate, kilobase-scale genome engineering applications.

Opioid-free anesthesia versus opioid-based anesthesia in patients undergoing cardiovascular and thoracic surgery: a meta-analysis and systematic review.

BACKGROUND: Despite their extensive clinical use, opioids are characterized by several side effects. These complications, coupled with the ongoing opioid epidemic, have favored the rise of opioid-free-anesthesia (OFA). Herein, we perform the first pairwise meta-analysis of clinical outcomes for OFA vs opioid-based anesthesia (OBA) in patients undergoing cardiovascular and thoracic surgery. METHODS: We comprehensively searched medical databases to identify studies comparing OFA and OBA in patients undergoing cardiovascular or thoracic surgery. Pairwise meta-analysis was performed using the Mantel-Haenszel method. Outcomes were pooled as risk ratios (RR) or standard mean differences (SMD) and their 95% confidence intervals (95% CI). RESULTS: Our pooled analysis included 919 patients (8 studies), of whom 488 underwent surgery with OBA and 431 with OFA. Among cardiovascular surgery patients, compared to OBA, OFA was associated with significantly reduced post-operative nausea and vomiting (RR, 0.57; P = .042), inotrope need (RR .84, P = .045), and non-invasive ventilation (RR, .54; P = .028). However, no differences were observed for 24hr pain score (SMD, -.35; P = .510) or 48hr morphine equivalent consumption (SMD, -1.09; P = .139). Among thoracic surgery patients, there was no difference between OFA and OBA for any of the explored outcomes, including post-operative nausea and vomiting (RR, 0.41; P = .025). CONCLUSION: Through the first pooled analysis of OBA vs OFA in a cardiothoracic-exclusive cohort, we found no significant difference in any of the pooled outcomes for thoracic surgery patients. Although limited to 2 cardiovascular surgery studies, OFA was associated with significantly reduced postoperative nausea and vomiting, inotrope need, and non-invasive ventilation in these patients. With growing use of OFA in invasive operations, further studies are needed to assess their efficacy and safety in cardiothoracic patients.

General Anesthesia Versus Local Anesthesia in Patients Undergoing Transcatheter Aortic Valve Replacement: An Updated Meta-Analysis and Systematic Review.

OBJECTIVES: For patients with aortic stenosis, transcatheter aortic valve replacement (TAVR) offers a less invasive treatment modality than conventional surgical valve replacement. Although the surgery is performed traditionally under general anesthesia (GA), recent studies have described success with TAVR using local anesthesia (LA) and/or conscious sedation. The study authors performed a pairwise meta-analysis to compare the clinical outcomes of TAVR based on operative anesthesia management. DESIGN: A random effects pairwise meta-analysis via the Mantel-Haenszel method. SETTING: Not applicable, as this is a meta-analysis. PARTICIPANTS: No individual patient data were used. INTERVENTIONS: Not applicable, as this is a meta-analysis. MEASUREMENTS AND MAIN RESULTS: The authors comprehensively searched the PubMed, Embase, and Cochrane databases to identify studies comparing TAVR performed using LA or GA. Outcomes were pooled as risk ratios (RR) or standard mean differences (SMD) and their 95% CIs. The authors' pooled analysis included 14,388 patients from 40 studies (7,754 LA; 6,634 GA). Compared to GA TAVR, LA TAVR was associated with significantly lower rates of 30-day mortality (RR 0.69; p < 0.01) and stroke (RR 0.78; p = 0.02). Additionally, LA TAVR patients had lower rates of 30-day major and/or life-threatening bleeding (RR 0.64; p = 0.01), 30-day major vascular complications (RR 0.76; p = 0.02), and long-term mortality (RR 0.75; p = 0.009). No significant difference was seen between the 2 groups for a 30-day paravalvular leak (RR 0.88, p = 0.12). CONCLUSIONS: Transcatheter aortic valve replacement performed using LA is associated with lower rates of adverse clinical outcomes, including 30-day mortality and stroke. No difference was seen between the 2 groups for a 30-day paravalvular leak. These results support the use of minimally invasive forms of TAVR without GA.

Selective TnsC recruitment enhances the fidelity of RNA-guided transposition.

Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site using TnsD1, whereas CRISPR-associated transposons use type I or type V Cas effectors to insert downstream of target sites specified by guide RNAs2,3. Despite this targeting diversity, transposition invariably requires TnsB, a DDE-family transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between transposase and targeting proteins4. How TnsC mediates this communication and thereby regulates transposition fidelity has remained unclear. Here we use chromatin immunoprecipitation with sequencing to monitor in vivo formation of the type I-F RNA-guided transpososome, enabling us to resolve distinct protein recruitment events before integration. DNA targeting by the TniQ-Cascade complex is surprisingly promiscuous-hundreds of genomic off-target sites are sampled, but only a subset of those sites is licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we determined structures of TnsC using cryogenic electron microscopy and found that ATP binding drives the formation of heptameric rings that thread DNA through the central pore, thereby positioning the substrate for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive factor binding to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts.

CD40/CD40L Signaling as a Promising Therapeutic Target for the Treatment of Renal Disease.

The cluster of differentiation 40 (CD40) is activated by the CD40 ligand (CD40L) in a variety of diverse cells types and regulates important processes associated with kidney disease. The CD40/CD40L signaling cascade has been comprehensively studied for its roles in immune functions, whereas the signaling axis involved in local kidney injury has only drawn attention in recent years. Clinical studies have revealed that circulating levels of soluble CD40L (sCD40L) are associated with renal function in the setting of kidney disease. Levels of the circulating CD40 receptor (sCD40), sCD40L, and local CD40 expression are tightly related to renal injury in different types of kidney disease. Additionally, various kidney cell types have been identified as non-professional antigen-presenting cells (APCs) that express CD40 on the cell membrane, which contributes to the interactions between immune cells and local kidney cells during the development of kidney injury. Although the potential for adverse CD40 signaling in kidney cells has been reported in several studies, a summary of those studies focusing on the role of CD40 signaling in the development of kidney disease is lacking. In this review, we describe the outcomes of recent studies and summarize the potential therapeutic methods for kidney disease which target CD40.

Bioorthogonal chemistry-based RNA labeling technologies: evolution and current state.

To understand the structure and ensuing function of RNA in various cellular processes, researchers greatly rely on traditional as well as contemporary labeling technologies to devise efficient biochemical and biophysical platforms. In this context, bioorthogonal chemistry based on chemoselective reactions that work under biologically benign conditions has emerged as a state-of-the-art labeling technology for functionalizing biopolymers. Implementation of this technology on sugar, protein, lipid and DNA is fairly well established. However, its use in labeling RNA has posed challenges due to the fragile nature of RNA. In this feature article, we provide an account of bioorthogonal chemistry-based RNA labeling techniques developed in our lab along with a detailed discussion on other technologies put forward recently. In particular, we focus on the development and applications of covalent methods to label RNA by transcription and posttranscription chemo-enzymatic approaches. It is expected that existing as well as new bioorthogonal functionalization methods will immensely advance our understanding of RNA and support the development of RNA-based diagnostic and therapeutic tools.

Responsive fluorescent nucleotides serve as efficient substrates to probe terminal uridylyl transferase.

We repurposed a terminal uridylyl transferase enzyme to site-specifically label RNA with microenvironment sensing fluorescent nucleotide mimics, which in turn provided direct read-outs to estimate the binding affinities of the enzyme to RNA and nucleotide substrates. This enzyme-probe system provides insights into the catalytic cycle, and can facilitate the development of discovery platforms to identify robust enzyme inhibitors.

Terminal Uridylyl Transferase Mediated Site-Directed Access to Clickable Chromatin Employing CRISPR-dCas9.

Locus-specific interrogation of target genes employing functional probes such as proteins and small molecules is paramount in decoding the molecular basis of gene function and designing tools to modulate its downstream effects. In this context, CRISPR-based gene editing and targeting technologies have proved tremendously useful, as they can be programmed to target any gene of interest by simply changing the sequence of the single guide RNA (sgRNA). Although these technologies are widely utilized in recruiting genetically encoded functional proteins, display of small molecules using CRISPR system is not well developed due to the lack of adequate techniques. Here, we have devised an innovative technology called sgRNA-Click (sgR-CLK) that harnesses the power of bioorthogonal click chemistry for remodeling guide RNA to display synthetic molecules on target genes. sgR-CLK employs a novel posttranscriptional chemoenzymatic labeling platform wherein a terminal uridylyl transferase (TUTase) was repurposed to generate clickable sgRNA of choice by site-specific tailoring of multiple azide-modified nucleotide analogues at the 3' end. The presence of a minimally invasive azide handle assured that the sgRNAs are indeed functional. Notably, an azide-tailed sgRNA targeting the telomeric repeat served as a Trojan horse on the CRISPR-dCas9 system to guide synthetic tags (biotin) site-specifically on chromatin employing copper-catalyzed or strain-promoted click reactions. Taken together, sgR-CLK presents a significant advancement on the utility of bioorthogonal chemistry, TUTase, and the CRISPR toolbox, which could offer a simplified solution for site-directed display of small molecule probes and diagnostic tools on target genes.

Vinyluridine as a Versatile Chemoselective Handle for the Post-transcriptional Chemical Functionalization of RNA.

The development of modular and efficient methods to functionalize RNA with biophysical probes is very important in advancing the understanding of the structural and functional relevance of RNA in various cellular events. Herein, we demonstrate a two-step bioorthogonal chemical functionalization approach for the conjugation of multiple probes onto RNA transcripts using a 5-vinyl-modified uridine nucleotide analog (VUTP). VUTP, containing a structurally noninvasive and versatile chemoselective handle, was efficiently incorporated into RNA transcripts by in vitro transcription reactions. Furthermore, we show for the first time the use of a palladium-mediated oxidative Heck reaction in functionalizing RNA with fluorogenic probes by reacting vinyl-labeled RNA transcripts with appropriate boronic acid substrates. The vinyl label also permitted the post-transcriptional functionalization of RNA by a reagent-free inverse electron demand Diels-Alder (IEDDA) reaction in the presence of tetrazine substrates. Collectively, our results demonstrate that the incorporation of VUTP provides newer possibilities for the modular functionalization of RNA with variety of reporters.

Posttranscriptional chemical labeling of RNA by using bioorthogonal chemistry.

Recent developments in RNA labeling technology have provided viable tools to analyze RNA synthesis, processing and function in cell-free and cellular environments. Notably, emerging methodologies based on posttranscriptional chemical labeling by using bioorthogonal chemistry have enabled the visualization and profiling of exogenous and endogenous RNA transcripts. In this review, we first give an overview of different RNA labeling strategies based on chemical as well as genetically encoded systems. Subsequently, we provided a detailed discussion on methodologies that have been developed to introduce various bioorthogonal reactive groups into RNA transcripts, which are compatible for posttranscriptional functionalization. Finally, the utility of these techniques in imaging and studying the dynamics of RNA production, distribution and decay in complex cellular environment is discussed.

Determination of Nicotine Content and Delivery in Disposable Electronic Cigarettes Available in the United States by Gas Chromatography-Mass Spectrometry.

INTRODUCTION: Electronic cigarettes (E-Cigs) are popular alternatives to conventional tobacco cigarettes. Disposable E-Cigs are single-use devices that emit aerosols from a nicotine-containing solution (e-liquid) by activating a heating coil during puffing. However, due to lack of regulations and standards, it is unclear how product claims are aligning with actual content and performance. Some analytical methods for characterizing E-Cigs are still in an exploratory phase. METHODS: Five products of disposable E-Cigs (purchased March-April, 2014 from a local smoke shop and an on-line US distributor) were studied for nicotine content, number of puffs obtained before depletion, portion of nicotine delivered via aerosolization, and e-liquid pH. Protocols were developed to consistently extract e-liquid from puffed and unpuffed E-Cigs. An in-house mechanical puffing machine was used to consistently puff E-Cig aerosols onto filter pads. A gas chromatography-mass spectrometry method was developed that produced sensitive and repeatable nicotine determinations. RESULTS: Under our experimental parameters, results showed a disparity between nicotine content and number of puffs achieved relative to what was claimed on product packaging. The portion of nicotine delivered to filter pads was often less than half that which was available, indicating much of the nicotine may be left in the E-Cig upon depletion. CONCLUSIONS: Analyses of unpuffed E-Cigs by gas chromatography-mass spectrometry indicate the nicotine content of these products can be considerably different from manufacture's labeling. Furthermore, a large portion of the nicotine in E-Cigs may not be transferred to the user, and that which is transferred, may often be in the less bioavailable form.

A base-modified PNA-graphene oxide platform as a turn-on fluorescence sensor for the detection of human telomeric repeats.

Given the biological and therapeutic significance of telomeres and other G-quadruplex forming sequences in human genome, it is highly desirable to develop simple methods to study these structures, which can also be implemented in screening formats for the discovery of G-quadruplex binders. The majority of telomere detection methods developed so far are laborious and use elaborate assay and instrumental setups, and hence, are not amenable to discovery platforms. Here, we describe the development of a simple homogeneous fluorescence turn-on method, which uses a unique combination of an environment-sensitive fluorescent nucleobase analogue, the superior base pairing property of PNA, and DNA-binding and fluorescence quenching properties of graphene oxide, to detect human telomeric DNA repeats of varying lengths. Our results demonstrate that this method, which does not involve a rigorous assay setup, would provide new opportunities to study G-quadruplex structures.