A novel 4,4-dimethylcytidine in the archaeal ribosome enhances hyperthermophily.

Ribosome structure and activity are challenged at high temperatures, often demanding modifications to ribosomal RNAs (rRNAs) to retain translation fidelity. LC-MS/MS, bisulfite-sequencing, and high-resolution cryo-EM structures of the archaeal ribosome identified an RNA modification, 4,4-dimethylcytidine (mC), at the universally conserved C918 in the 16S rRNA helix 31 loop. Here, we characterize and structurally resolve a class of RNA methyltransferase that generates mC whose function is critical for hyperthermophilic growth.

Structural basis of archaeal FttA-dependent transcription termination.

The ribonuclease FttA (also known as aCPSF and aCPSF1) mediates factor-dependent transcription termination in archaea. Here we report the structure of a Thermococcus kodakarensis transcription pre-termination complex comprising FttA, Spt4, Spt5 and a transcription elongation complex (TEC). The structure shows that FttA interacts with the TEC in a manner that enables RNA to proceed directly from the TEC RNA-exit channel to the FttA catalytic centre and that enables endonucleolytic cleavage of RNA by FttA, followed by 5'→3' exonucleolytic cleavage of RNA by FttA and concomitant 5'→3' translocation of FttA on RNA, to apply mechanical force to the TEC and trigger termination.

Intein splicing efficiency and RadA levels can control the mode of archaeal DNA replication.

Inteins (intervening proteins), mobile genetic elements removed through protein splicing, often interrupt proteins required for DNA replication, recombination, and repair. An abundance of in vitro evidence implies that inteins may act as regulatory elements, whereby reduced splicing inhibits production of the mature protein lacking the intein, but in vivo evidence of regulatory intein excision in the native host is absent. The model archaeon encodes 15 inteins, and we establish the impacts of intein splicing inhibition on host physiology and replication in vivo.

The extensive mC epitranscriptome of Thermococcus kodakarensis is generated by a suite of RNA methyltransferases that support thermophily.

RNAs are often modified to invoke new activities. While many modifications are limited in frequency, restricted to non-coding RNAs, or present only in select organisms, 5-methylcytidine (mC) is abundant across diverse RNAs and fitness-relevant across Domains of life, but the synthesis and impacts of mC have yet to be fully investigated. Here, we map mC in the model hyperthermophile, Thermococcus kodakarensis.

Archaeal histone-based chromatin structures regulate transcription elongation rates.

Many archaea encode and express histone proteins to compact their genomes. Archaeal and eukaryotic histones share a near-identical fold that permits DNA wrapping through select histone-DNA contacts to generate chromatin-structures that must be traversed by RNA polymerase (RNAP) to generate transcripts. As archaeal histones can spontaneously assemble with a single histone isoform, single-histone chromatin variants provide an idealized platform to detail the impacts of distinct histone-DNA contacts on transcription efficiencies and to detail the role of the conserved cleavage stimulatory factor, Transcription Factor S (TFS), in assisting RNAP through chromatin landscapes.

Tetraether archaeal lipids promote long-term survival in extreme conditions.

The sole unifying feature of the incredibly diverse Archaea is their isoprenoid-based ether-linked lipid membranes. Unique lipid membrane composition, including an abundance of membrane-spanning tetraether lipids, impart resistance to extreme conditions. Many questions remain, however, regarding the synthesis and modification of tetraether lipids and how dynamic changes to archaeal lipid membrane composition support hyperthermophily.

Thermococcus kodakarensis TK0353 is a novel AP lyase with a new fold.

Hyperthermophilic organisms thrive in extreme environments prone to high levels of DNA damage. Growth at high temperature stimulates DNA base hydrolysis resulting in apurinic/apyrimidinic (AP) sites that destabilize the genome. Organisms across all domains have evolved enzymes to recognize and repair AP sites to maintain genome stability.

Structural basis of archaeal FttA-dependent transcription termination.

Unlabelled: The ribonuclease FttA mediates factor-dependent transcription termination in archaea . Here, we report the structure of a transcription pre-termination complex comprising FttA, Spt4, Spt5, and a transcription elongation complex (TEC). The structure shows that FttA interacts with the TEC in a manner that enables RNA to proceed directly from the TEC RNA-exit channel to the FttA catalytic center and that enables endonucleolytic cleavage of RNA by FttA, followed by 5'→3' exonucleolytic cleavage of RNA by FttA and concomitant 5'→3' translocation of FttA on RNA, to apply mechanical force to the TEC and trigger termination.

Histones direct site-specific CRISPR spacer acquisition in model archaeon.

CRISPR-Cas systems provide heritable immunity against viruses and other mobile genetic elements by incorporating fragments of invader DNA into the host CRISPR array as spacers. Integration of new spacers is localized to the 5' end of the array, and in certain Gram-negative Bacteria this polarized localization is accomplished by the integration host factor. For most other Bacteria and Archaea, the mechanism for 5' end localization is unknown.

Transformation Techniques for the Anaerobic Hyperthermophile Thermococcus kodakarensis.

Genetic manipulation is an essential tool to investigate complex microbiological phenomena. In this chapter we describe the techniques required to transform the model hyperthermophilic, anaerobic archaeon Thermococcus kodakarensis. T.

The structure and activities of the archaeal transcription termination factor Eta detail vulnerabilities of the transcription elongation complex.

Transcription must be properly regulated to ensure dynamic gene expression underlying growth, development, and response to environmental cues. Regulation is imposed throughout the transcription cycle, and while many efforts have detailed the regulation of transcription initiation and early elongation, the termination phase of transcription also plays critical roles in regulating gene expression. Transcription termination can be driven by only a few proteins in each domain of life.

Compressed Sensing Cardiac Cine Imaging Compared with Standard Balanced Steady-State Free Precession Cine Imaging in a Pediatric Population.

Purpose: To compare real-time compressed sensing (CS) and standard balanced steady-state free precession (bSSFP) cardiac cine imaging in children.

Materials And Methods: Twenty children (mean age, 15 years ± 5 [SD], range, 7-21 years; 10 male participants) with biventricular congenital heart disease ( = 11) or cardiomyopathy ( = 9) were prospectively included. Examinations were performed with 1.

Changes in post-treatment cardiac PET avidity predict overall survival in lung cancer patients treated with chemoradiation: Secondary analysis of the ACRIN 6668/RTOG 0235 clinical trial.

The purpose of this work was to use data from a large co-operative group trial to evaluate whether metabolic FDG-PET changes in the heart for lung cancer patients can predict for clinical outcomes. The study found that cardiac SUV changes following definitive chemoradiation are significantly (HR 0.811, 95% CI 0.

State-of-the-art review of lung imaging in cystic fibrosis with recommendations for pulmonologists and radiologists from the "iMAging managEment of cySTic fibROsis" (MAESTRO) consortium.

Objective: Imaging represents an important noninvasive means to assess cystic fibrosis (CF) lung disease, which remains the main cause of morbidity and mortality in CF patients. While the development of new imaging techniques has revolutionised clinical practice, advances have posed diagnostic and monitoring challenges. The authors aim to summarise these challenges and make evidence-based recommendations regarding imaging assessment for both clinicians and radiologists.

Thermococcus kodakarensis provides a versatile hyperthermophilic archaeal platform for protein expression.

Hyperthermophiles, typically defined as organisms with growth optima ≥80°C, are dominated by the Archaea. Proteins that support life at the extremes of temperatures often retain substantial biotechnological and commercial value, but the recombinant expression of individual hyperthermophilic proteins is commonly complicated in non-native mesophilic hosts due to differences in codon bias, intracellular solutes and the requirement for accessory factors that aid in folding or deposition of metal centers within archaeal proteins. The development of versatile protein expression and facilitated protein purification systems in the model, genetically tractable, hyperthermophilic marine archaeon Thermococcus kodakarensis provides an attractive platform for protein expression within the hyperthermophiles.

The Role of Lung Function Testing in Newborn Infants With Congenital Thoracic Arterial Anomalies.

Congenital thoracic arterial anomalies (CTAAs), such as complete or incomplete vascular rings, pulmonary artery sling, and innominate artery compression syndrome, may cause severe tracheomalacia and upper airway obstruction. An obstructive ventilatory pattern at lung function testing (LFT) has been suggested in the presence of CTAA. The severity of obstruction may be evaluated by LFT.

The Hyperthermophilic Restriction-Modification Systems of Protect Genome Integrity.

(), a hyperthermophilic, genetically accessible model archaeon, encodes two putative restriction modification (R-M) defense systems, TkoI and TkoII. TkoI is encoded by TK1460 while TkoII is encoded by TK1158. Bioinformative analysis suggests both R-M enzymes are large, fused methyltransferase (MTase)-endonuclease polypeptides that contain both restriction endonuclease (REase) activity to degrade foreign invading DNA and MTase activity to methylate host genomic DNA at specific recognition sites.

Extended Archaeal Histone-Based Chromatin Structure Regulates Global Gene Expression in .

Histone proteins compact and organize DNA resulting in a dynamic chromatin architecture impacting DNA accessibility and ultimately gene expression. Eukaryotic chromatin landscapes are structured through histone protein variants, epigenetic marks, the activities of chromatin-remodeling complexes, and post-translational modification of histone proteins. In most Archaea, histone-based chromatin structure is dominated by the helical polymerization of histone proteins wrapping DNA into a repetitive and closely gyred configuration.

Tracheal trifurcation: new cases and review of the literature.

Background: Tracheal trifurcation is an uncommon and often unknown type of anomalous tracheobronchial arborization, characterized by three main bronchi originating at the level of the carina. Diagnosis is important due to its clinical implications.

Objective: To highlight the anatomical, clinical and diagnostic aspects of tracheal trifurcation by reporting our experience and reviewing the literature.

Blood flow characteristics after aortic valve neocuspidization in paediatric patients: a comparison with the Ross procedure.

Aims: The aortic valve (AV) neocuspidization (Ozaki procedure) is a novel surgical technique for AV disease that preserves the natural motion and cardiodynamics of the aortic root. In this study, we sought to evaluate, by 4D-flow magnetic resonance imaging, the aortic blood flow characteristics after AV neocuspidization in paediatric patients.

Methods And Results: Aortic root and ascending aorta haemodynamics were evaluated in a population of patients treated with the Ozaki procedure; results were compared with those of a group of patients operated with the Ross technique.

Dynamic contrast-enhanced magnetic resonance lymphangiography in pediatric patients with central lymphatic system disorders.

Central conducting lymphatics (CCLs) disorders represent a broad spectrum of clinical entities ranging from self-limiting traumatic leaks treated by conservative strategies, to complex lymphatic circulation abnormalities that are progressive and unresponsive to currently available treatments. Dynamic contrast-enhanced magnetic resonance lymphangiography (DCMRL) performed by intranodal injection of gadolinium-based contrast material is a recently developed technique which allows a minimally invasive evaluation of the CCL abnormalities providing a dynamic assessment of lymph flow and its pathways. In our institution, DCMRL is performed after bilateral cannulation of inguinal lymph nodes, using a MR protocol which includes volumetric 3D T2-SPACE (sampling perfection with application-optimized contrasts using different flip-angle evolution) and free-breathing respiratory navigated sequence and TWIST (time-resolved angiography with Interleaved stochastic trajectories) MR angiography sequence, during intranodal injection of paramagnetic contrast medium.

Archaeal DNA Repair Mechanisms.

Archaea often thrive in environmental extremes, enduring levels of heat, pressure, salinity, pH, and radiation that prove intolerable to most life. Many environmental extremes raise the propensity for DNA damaging events and thus, impact DNA stability, placing greater reliance on molecular mechanisms that recognize DNA damage and initiate accurate repair. Archaea can presumably prosper in harsh and DNA-damaging environments in part due to robust DNA repair pathways but surprisingly, no DNA repair pathways unique to Archaea have been described.

Archaeal transcription.

Increasingly sophisticated biochemical and genetic techniques are unraveling the regulatory factors and mechanisms that control gene expression in the Archaea. While some similarities in regulatory strategies are universal, archaeal-specific regulatory strategies are emerging to complement a complex patchwork of shared archaeal-bacterial and archaeal-eukaryotic regulatory mechanisms employed in the archaeal domain. The prokaryotic archaea encode core transcription components with homology to the eukaryotic transcription apparatus and also share a simplified eukaryotic-like initiation mechanism, but also deploy tactics common to bacterial systems to regulate promoter usage and influence elongation-termination decisions.