Localization of RNAs to the mitochondria-mechanisms and functions.

The mammalian mitochondrial proteome comprises over 1000 proteins, with the majority translated from nuclear-encoded messenger RNAs (mRNAs). Mounting evidence suggests many of these mRNAs are localized to the outer mitochondrial membrane (OMM) in a pre- or cotranslational state. Upon reaching the mitochondrial surface, these mRNAs are locally translated to produce proteins that are cotranslationally imported into mitochondria. Here, we summarize various mechanisms cells use to localize RNAs, including transfer RNAs (tRNAs), to the OMM and recent technological advancements in the field to study these processes. While most early studies in the field were carried out in yeast, recent studies reveal RNA localization to the OMM and their regulation in higher organisms. Various factors regulate this localization process, including RNA sequence elements, RNA-binding proteins (RBPs), cytoskeletal motors, and translation machinery. In this review, we also highlight the role of RNA structures and modifications in mitochondrial RNA localization and discuss how these features can alter the binding properties of RNAs. Finally, in addition to RNAs related to mitochondrial function, RNAs involved in other cellular processes can also localize to the OMM, including those implicated in the innate immune response and piRNA biogenesis. As impairment of messenger RNA (mRNA) localization and regulation compromise mitochondrial function, future studies will undoubtedly expand our understanding of how RNAs localize to the OMM and investigate the consequences of their mislocalization in disorders, particularly neurodegenerative diseases, muscular dystrophies, and cancers.

The role of chromium supplementation in cardiovascular risk factors: A comprehensive reviews of putative molecular mechanisms.

In the recent years, micronutrients play an important role in improving body health with preventing and treating of chronic diseases. Chromium is one of the vital minerals involved in the regulation of insulin action. According to abundant evidences this mineral seems to be an essential factor involved in the reduction of insulin resistance and decreasing the risk of type 2 diabetes mellitus (T2DM) and cardiovascular diseases (CVDs). Moreover, it has been proposed that Chromium supplementation affects mechanisms involved in blood pressure, lipid metabolism, inflammation, and oxidative stress. For instance, it may affect blood pressure through alteration of the renin-angiotensin system, as well as reducing the angiotensin-converting enzyme activity. Furthermore, Chromium supplementation might help reduce the coronary heart disease rates. This study aims to provide a comprehensive review regarding to the effects of Chromium supplementation on CVDs risk factors with an emphasis on possible molecular mechanisms.

Avasopasem manganese (GC4419) protects against cisplatin-induced chronic kidney disease: An exploratory analysis of renal metrics from a randomized phase 2b clinical trial in head and neck cancer patients.

Head and neck squamous cell carcinoma (HNSCC) patients treated with high-dose cisplatin concurrently with radiotherapy (hdCis-RT) commonly suffer kidney injury leading to acute and chronic kidney disease (AKD and CKD, respectively). We conducted a retrospective analysis of renal function and kidney injury-related plasma biomarkers in a subset of HNSCC subjects receiving hdCis-RT in a double-blinded, placebo-controlled clinical trial (NCT02508389) evaluating the superoxide dismutase mimetic, avasopasem manganese (AVA), an investigational new drug. We found that 90 mg AVA treatment prevented a significant reduction in estimated glomerular filtration rate (eGFR) three months as well as six and twelve months after treatment compared to 30 mg AVA and placebo. Moreover, AVA treatment may have allowed renal repair in the first 22 days following cisplatin treatment as evidenced by an increase in epithelial growth factor (EGF), known to aid in renal recovery. An upward trend was also observed in plasma iron homeostasis proteins including total iron (Fe-blood) and iron saturation (Fe-saturation) in the 90 mg AVA group versus placebo. These data support the hypothesis that treatment with 90 mg AVA mitigates cisplatin-induced CKD by inhibiting hdCis-induced renal changes and promoting renal recovery.

Phase Ib study of eprenetapopt (APR-246) in combination with pembrolizumab in patients with advanced or metastatic solid tumors.

BACKGROUND: We conducted a phase I, multicenter, open-label, dose-finding, and expansion study to determine the safety and preliminary efficacy of eprenetapopt (APR-246) combined with pembrolizumab in patients with advanced/metastatic solid tumors (ClinicalTrials.gov NCT04383938). PATIENTS AND METHODS: For dose-finding, requirements were non-central nervous system primary solid tumor, intolerant to/progressed after ≥1 line of treatment, and eligible for pembrolizumab; for expansion: (i) gastric/gastroesophageal junction tumor, intolerant to/progressed after first-line treatment, and no prior anti-programmed cell death receptor-1 (PD-1)/programmed death-ligand 1 (PD-L1) therapy; (ii) bladder/urothelial tumor, intolerant to/progressed after first-line cisplatin-based chemotherapy, and no prior anti-PD-1/PD-L1 therapy; (iii) non-small-cell lung cancer (NSCLC) with previous anti-PD-1/PD-L1 therapy. Patients received eprenetapopt 4.5 g/day intravenously (IV) on days 1-4 with pembrolizumab 200 mg IV on day 3 in each 21-day cycle. Primary endpoints were dose-limiting toxicity (DLT), adverse events (AEs), and recommended phase II dose (RP2D) of eprenetapopt. RESULTS: Forty patients were enrolled (median age 66 years; range 27-85) and 37 received eprenetapopt plus pembrolizumab. No DLTs were reported and the RP2D for eprenetapopt in combination was 4.5 g/day IV on days 1-4. The most common eprenetapopt-related AEs were dizziness (35.1%), nausea (32.4%), and vomiting (29.7%). AEs leading to eprenetapopt discontinuation occurred in 2/37 patients (5.4%). In efficacy-assessable patients (n = 29), one achieved complete response (urothelial cancer), two achieved partial responses (NSCLC, urothelial cancer), and six patients had stable disease. CONCLUSIONS: The eprenetapopt plus pembrolizumab combination was well tolerated with an acceptable safety profile and showed clinical activity in patients with solid tumors.

Parents' Experience of Having an Infant in the Neonatal Intensive Care Unit: A Qualitative Study.

INTRODUCTION: Admission to the neonatal intensive care unit (NICU) is usually unexpected and can be stressful to the parents causing strenuous psychosocial effects. Parents of these infants are subject to suffering stress, depression, and feelings of powerlessness. This study aimed at describing parents' experience of having their infant in the neonatal intensive care unit. METHOD:  A qualitative descriptive design was used. Parents (six couples and four mothers) of infants hospitalized for at least ten days regardless of gestational age, gender, or medical diagnosis were selected from a teaching hospital in Amman, Jordan. Semi-structured interviews were conducted between June 2019 and November 2019. RESULTS:  Thematic analysis of the data revealed four emerging themes: (1) Living the ambiguities of the admission to the NICU, (2) Living the burdens of their infants' hospitalization, (3) Coping with the stresses of a hospitalized infant, and (4) Reflecting on interactions with healthcare staff and the environment. DISCUSSION AND CONCLUSION:  The study findings demonstrated parents' worries and needs and highlighted the use of spirituality/religiosity as a coping mechanism. The findings will guide healthcare providers and policymakers to develop caring strategies that enhance care delivered to parents of infants in intensive care units.

Invariant natural killer T-cell subsets have diverse graft-versus-host-disease-preventing and antitumor effects.

Invariant natural killer T (iNKT) cells are a T-cell subset with potent immunomodulatory properties. Experimental evidence in mice and observational studies in humans indicate that iNKT cells have antitumor potential as well as the ability to suppress acute and chronic graft-versus-host-disease (GVHD). Murine iNKT cells differentiate during thymic development into iNKT1, iNKT2, and iNKT17 sublineages, which differ transcriptomically and epigenomically and have subset-specific developmental requirements. Whether distinct iNKT sublineages also differ in their antitumor effect and their ability to suppress GVHD is currently unknown. In this work, we generated highly purified murine iNKT sublineages, characterized their transcriptomic and epigenomic landscape, and assessed specific functions. We show that iNKT2 and iNKT17, but not iNKT1, cells efficiently suppress T-cell activation in vitro and mitigate murine acute GVHD in vivo. Conversely, we show that iNKT1 cells display the highest antitumor activity against murine B-cell lymphoma cells both in vitro and in vivo. Thus, we report for the first time that iNKT sublineages have distinct and different functions, with iNKT1 cells having the highest antitumor activity and iNKT2 and iNKT17 cells having immune-regulatory properties. These results have important implications for the translation of iNKT cell therapies to the clinic for cancer immunotherapy as well as for the prevention and treatment of GVHD.

Effectiveness of phytase pre-treatment on growth performance, nutrient digestibility and mineral status of common carp (Cyprinus carpio) juveniles fed Moringa by-product based diet.

Several anti-nutritional substances are found in plant derivatives for example phytate, that make the nutrients and minerals unavailable to fish, hence leading to poor growth performance. Presence of the anti-nutrient factor such as phytate is a chelated compound and need enzyme for its breakdown and availability of nutrients to improve fish growth. This research work was performed to check the improvement of overall performance of Cyprinus carpio fingerlings by the help of phytase addition in Moringa oleifera by- products based diet. Combination of Moringa seed meal and Moringa leaf meal was utilized as test ingredient to formulate seven test feeds, containing graded levels of phytase (0, 500, 650, 800, 950, 1100 and 1250 FTU kg-1). In feeding trial of 70 days, fingerlings were given feed two times in a day at the rate of 4% of wet weight of their bodies and faeces were collected. According to current results, it was found that growth performance parameters i.e. weight gain; 25 g, specific growth rate; 1.67 and feed conversion ratio; 1.10 were improved to maximum at 950 FTU kg-1. Digestibility of nutrients (crude protein; 73%, crude fat; 71% and gross energy; 67%) and minerals absorption was also maximum (Ca; 70%, Zn; 66%, K; 74%, Mn; 66% and P; 71%) at 950 FTU kg-1. Lowest growth efficiency, nutrient digestibility and mineral absorption were observed in fingerlings fed at control diet (0 FTU kg-1). Results of the current study, proved that 950 FTU kg-1 is the most optimum level of phytase to formulate economical and ecofriendly feed for improved growth of C. carpio fingerlings as it decreases the discharge of minerals and nutrients in water bodies.

Incidence of septicemia. Etiology and antimicrobial susceptibility testing among patients admitted to tertiary care hospital.

INTRODUCTION: Septicemia is considered as an important cause of life-threating infections. The study was aimed at determining the incidence of septicemia considering different age groups and gender among suspected patients admitted to a tertiary care hospital in Iraq. METHODOLOGY: A total of 168 blood samples were collected and cultured using BacT/Alert 3D automated system. The isolated pathogens were identified and subjected to antimicrobial susceptibility testing using automated Vitek 2 Compact system. RESULTS: Out of 168 blood samples, 53 (31.5%) gave positive microbial growth. Thirty-three samples (62.3%) came from male patients and 20 (37.7%) from female ones, both gender and microbial growth were significantly related (P < 0.05). Age group (21 year - 30 year) was found to have the highest percentage of positive growth (26.4%) while age group (51 year - 60 year) the lowest percentage (5.7%) of positive growth. Both microbial growth and age group were found to be associated to a significant level (P < 0.05). 36 isolates (67.9%) were Gram negative, 15 isolates (28.3%) were Gram-positive and 2 isolates (3.8%) were fungi. Salmonella typhi (41.7%) represented the most common pathogen isolated followed by Acinetobacter baumannii (22.2%). An isolate of Pseudomonas aeruginosa showed resistance to all antibiotics used. CONCLUSION: Community-acquired septicemia occurred mainly in male than female. Salmonella typhi and Acinetobacter baumannii represented the most frequent causative agents of community-acquired septicemia. Antimicrobial susceptibility testing should be performed to detect the antibiotic of choice for each pathogen causing community-acquired septicemia.

RNA-GPS Predicts SARS-CoV-2 RNA Residency to Host Mitochondria and Nucleolus.

SARS-CoV-2 genomic and subgenomic RNA (sgRNA) transcripts hijack the host cell's machinery. Subcellular localization of its viral RNA could, thus, play important roles in viral replication and host antiviral immune response. We perform computational modeling of SARS-CoV-2 viral RNA subcellular residency across eight subcellular neighborhoods. We compare hundreds of SARS-CoV-2 genomes with the human transcriptome and other coronaviruses. We predict the SARS-CoV-2 RNA genome and sgRNAs to be enriched toward the host mitochondrial matrix and nucleolus, and that the 5' and 3' viral untranslated regions contain the strongest, most distinct localization signals. We interpret the mitochondrial residency signal as an indicator of intracellular RNA trafficking with respect to double-membrane vesicles, a critical stage in the coronavirus life cycle. Our computational analysis serves as a hypothesis generation tool to suggest models for SARS-CoV-2 biology and inform experimental efforts to combat the virus. A record of this paper's Transparent Peer Review process is included in the Supplemental Information.

RNA-GPS predicts high-resolution RNA subcellular localization and highlights the role of splicing.

Subcellular localization is essential to RNA biogenesis, processing, and function across the gene expression life cycle. However, the specific nucleotide sequence motifs that direct RNA localization are incompletely understood. Fortunately, new sequencing technologies have provided transcriptome-wide atlases of RNA localization, creating an opportunity to leverage computational modeling. Here we present RNA-GPS, a new machine learning model that uses nucleotide-level features to predict RNA localization across eight different subcellular locations-the first to provide such a wide range of predictions. RNA-GPS's design enables high-throughput sequence ablation and feature importance analyses to probe the sequence motifs that drive localization prediction. We find localization informative motifs to be concentrated on 3'-UTRs and scattered along the coding sequence, and motifs related to splicing to be important drivers of predicted localization, even for cytotopic distinctions for membraneless bodies within the nucleus or for organelles within the cytoplasm. Overall, our results suggest transcript splicing is one of many elements influencing RNA subcellular localization.

Atlas of Subcellular RNA Localization Revealed by APEX-Seq.

We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.

RNA structure maps across mammalian cellular compartments.

RNA structure is intimately connected to each step of gene expression. Recent advances have enabled transcriptome-wide maps of RNA secondary structure, called 'RNA structuromes'. However, previous whole-cell analyses lacked the resolution to unravel the landscape and also the regulatory mechanisms of RNA structural changes across subcellular compartments. Here we reveal the RNA structuromes in three compartments, chromatin, nucleoplasm and cytoplasm, in human and mouse cells. The cytotopic structuromes substantially expand RNA structural information and enable detailed investigation of the central role of RNA structure in linking transcription, translation and RNA decay. We develop a resource with which to visualize the interplay of RNA-protein interactions, RNA modifications and RNA structure and predict both direct and indirect reader proteins of RNA modifications. We also validate a novel role for the RNA-binding protein LIN28A as an N6-methyladenosine modification 'anti-reader'. Our results highlight the dynamic nature of RNA structures and its functional importance in gene regulation.

Subcellular Spatial Transcriptomes: Emerging Frontier for Understanding Gene Regulation.

RNAs are trafficked and localized with exquisite precision inside the cell. Studies of candidate messenger RNAs have shown the vital importance of RNA subcellular location in development and cellular function. New sequencing- and imaging-based methods are providing complementary insights into subcellular localization of RNAs transcriptome-wide. APEX-seq and ribosome profiling as well as proximity-labeling approaches have revealed thousands of transcript isoforms are localized to distinct cytotopic locations, including locations that defy biochemical fractionation and hence were missed by prior studies. Sequences in the 3' and 5' untranslated regions (UTRs) serve as "zip codes" to direct transcripts to particular locales, and it is clear that intronic and retrotransposable sequences within transcripts have been co-opted by cells to control localization. Molecular motors, nuclear-to-cytosol RNA export, liquid-liquid phase separation, RNA modifications, and RNA structure dynamically shape the subcellular transcriptome. Location-based RNA regulation continues to pose new mysteries for the field, yet promises to reveal insights into fundamental cell biology and disease mechanisms.

Triple Antithrombotic Therapy for Patients with Atrial Fibrillation Undergoing Percutaneous Coronary Intervention.

Dual antiplatelet therapy (DAPT) has been the cornerstone of antithrombotic management for patients undergoing percutaneous coronary intervention (PCI). However, approximately 10% of these patients have concomitant atrial fibrillation (AF) and require chronic oral anticoagulant (OAC) in addition to DAPT. This traditional "triple therapy" has been associated with a three to four-fold increased risk of bleeding. The safety of non-vitamin K OAC (NOAC)-based strategies, using a NOAC plus a P2Y12 inhibitor, has been compared to vitamin K antagonist (VKA)-based triple therapy in the PIONEER AF-PCI and REDUAL PCI randomized trials, both of which have demonstrated that NOAC-based strategies are safer and provide an attractive alternative to VKA-based triple therapy among AF patients who undergo PCI. This article reviews the rationale, evidence, and recent evaluation of triple antithrombotic therapy among AF patients undergoing PCI.

Real-time observation of polymerase-promoter contact remodeling during transcription initiation.

Critical contacts made between the RNA polymerase (RNAP) holoenzyme and promoter DNA modulate not only the strength of promoter binding, but also the frequency and timing of promoter escape during transcription. Here, we describe a single-molecule optical-trapping assay to study transcription initiation in real time, and use it to map contacts formed between σ70 RNAP holoenzyme from E. coli and the T7A1 promoter, as well as to observe the remodeling of those contacts during the transition to the elongation phase. The strong binding contacts identified in certain well-known promoter regions, such as the -35 and -10 elements, do not necessarily coincide with the most highly conserved portions of these sequences. Strong contacts formed within the spacer region (-10 to -35) and with the -10 element are essential for initiation and promoter escape, respectively, and the holoenzyme releases contacts with promoter elements in a non-sequential fashion during escape.

lncRNA Structure: Message to the Heart.

In this issue, Xue et al. (2016) describe the secondary structure of the heart-specific long non-coding RNA Braveheart, leading to the discovery of a short, asymmetric G-rich loop that controls cardiac lineage commitment by interacting with the transcription factor CNBP.

Direct observation of processive exoribonuclease motion using optical tweezers.

Bacterial RNases catalyze the turnover of RNA and are essential for gene expression and quality surveillance of transcripts. In Escherichia coli, the exoribonucleases RNase R and polynucleotide phosphorylase (PNPase) play critical roles in degrading RNA. Here, we developed an optical-trapping assay to monitor the translocation of individual enzymes along RNA-based substrates. Single-molecule records of motion reveal RNase R to be highly processive: one molecule can unwind over 500 bp of a structured substrate. However, enzyme progress is interrupted by pausing and stalling events that can slow degradation in a sequence-dependent fashion. We found that the distance traveled by PNPase through structured RNA is dependent on the A+U content of the substrate and that removal of its KH and S1 RNA-binding domains can reduce enzyme processivity without affecting the velocity. By a periodogram analysis of single-molecule records, we establish that PNPase takes discrete steps of six or seven nucleotides. These findings, in combination with previous structural and biochemical data, support an asymmetric inchworm mechanism for PNPase motion. The assay developed here for RNase R and PNPase is well suited to studies of other exonucleases and helicases.

Real-time observation of the initiation of RNA polymerase II transcription.

Biochemical and structural studies have shown that the initiation of RNA polymerase II transcription proceeds in the following stages: assembly of the polymerase with general transcription factors and promoter DNA in a 'closed' preinitiation complex (PIC); unwinding of about 15 base pairs of the promoter DNA to form an 'open' complex; scanning downstream to a transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. Here we have assembled a 32-protein, 1.5-megadalton PIC derived from Saccharomyces cerevisiae, and observe subsequent initiation processes in real time with optical tweezers. Contrary to expectation, scanning driven by the transcription factor IIH involved the rapid opening of an extended transcription bubble, averaging 85 base pairs, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.