Mechanisms used for cDNA synthesis and site-specific integration of RNA into DNA genomes by a reverse transcriptase-Cas1 fusion protein.

Reverse transcriptase-Cas1 (RT-Cas1) fusion proteins found in some CRISPR systems enable spacer acquisition from both RNA and DNA, but the mechanism of RNA spacer acquisition has remained unclear. Here, we found that Marinomonas mediterranea RT-Cas1/Cas2 adds short 3'-DNA (dN) tails to RNA protospacers, enabling their direct integration into CRISPR arrays as 3'-dN-RNAs or 3'-dN-RNA/cDNA duplexes at rates comparable to similarly configured DNAs. Reverse transcription of RNA protospacers is initiated at 3' proximal sites by multiple mechanisms, including recently described de novo initiation, protein priming with any dNTP, and use of short exogenous or synthesized DNA oligomer primers, enabling synthesis of near full-length cDNAs of diverse RNAs without fixed sequence requirements. The integration of 3'-dN-RNAs or single-stranded DNAs (ssDNAs) is favored over duplexes at higher protospacer concentrations, potentially relevant to spacer acquisition from abundant pathogen RNAs or ssDNA fragments generated by phage defense nucleases. Our findings reveal mechanisms for site-specifically integrating RNA into DNA genomes with potential biotechnological applications.

The clinical presentation of avoidant restrictive food intake disorder in children and adolescents is largely independent of sex, autism spectrum disorder and anxiety traits.

Background: Avoidant restrictive food intake disorder (ARFID) is a new eating disorder with a heterogeneous clinical presentation. It is unclear which patient characteristics contribute to its heterogeneity. Methods: To identify these patient characteristics, we performed symptom-level correlation and driver-level regression analyses in our cross-sectional study in up to 261 ARFID patients (51% female; median age = 12.7 years) who were assessed at the Maudsley Centre for Child and Adolescent Eating Disorders, London between November 2019 and July 2022. Findings: Symptoms across the three drivers 1) avoidance based on sensory characteristics of food; 2) apparent lack of interest in eating; and 3) concern about aversive consequences positively correlated with each other. Patients' anxiety traits showed the greatest positive correlations with symptoms of concern about aversive consequences of eating. Patient sex was not significantly associated with any of the three ARFID drivers. Patients with comorbid autism spectrum disorder (ASD; 28%) showed more food-related sensory sensitivities (RR = 1.26) and greater lack of interest in eating (RR = 1.18) than those of patients without ASD (49%). Interpretation: In our clinical sample, the ARFID drivers occurred together and did not show clinically meaningful differences between the sexes. ASD may accentuate food-related sensory sensitivities and lack of interest, but may not drive a completely different symptom presentation. ARFID is multi-faceted and heterogenous, requiring a comprehensive multidisciplinary assessment to sufficiently understand the drivers of the restrictive eating behaviour. Results need replication in larger samples with more statistical power. Funding: None.

Mechanisms used for cDNA synthesis and site-specific integration of RNA into DNA genomes by a reverse transcriptase-Cas1 fusion protein.

Reverse transcriptase-Cas1 (RT-Cas1) fusion proteins found in some CRISPR systems enable spacer acquisition from both RNA and DNA, but the mechanism of RNA spacer acquisition has remained unclear. Here, we found Marinomonas mediterranea RT-Cas1/Cas2 adds short 3'-DNA (dN) tails to RNA protospacers enabling their direct integration into CRISPR arrays as 3'-dN-RNA/cDNA duplexes or 3'-dN-RNAs at rates comparable to similarly configured DNAs. Reverse transcription of RNA protospacers occurs by multiple mechanisms, including recently described de novo initiation, protein priming with any dNTP, and use of short exogenous or synthesized DNA oligomer primers, enabling synthesis of cDNAs from diverse RNAs without fixed sequence requirements. The integration of 3'-dN-RNAs or single-stranded (ss) DNAs is favored over duplexes at higher protospacer concentrations, potentially relevant to spacer acquisition from abundant pathogen RNAs or ssDNA fragments generated by phage-defense nucleases. Our findings reveal novel mechanisms for site-specifically integrating RNA into DNA genomes with potential biotechnological applications.

Validity and repeatability of cardiopulmonary exercise testing in interstitial lung disease.

BACKGROUND: Cardiopulmonary exercise testing (CPET), and its primary outcome of peak oxygen uptake (VO2peak), are acknowledged as biomarkers in the diagnostic and prognostic management of interstitial lung disease (ILD). However, the validity and repeatability of CPET in those with ILD has yet to be fully characterised, and this study fills this evidence gap. METHODS: Twenty-six people with ILD were recruited, and 21 successfully completed three CPETs. Of these, 17 completed two valid CPETs within a 3-month window, and 11 completed two valid CPETs within a 6-month window. Technical standards from the European Respiratory Society established validity, and repeatability was determined using mean change, intraclass correlation coefficient and typical error. RESULTS: Every participant (100%) who successfully exercised to volitional exhaustion produced a maximal, and therefore valid, CPET. Approximately 20% of participants presented with a plateau in VO2, the primary criteria for establishing a maximal effort. The majority of participants otherwise presented with secondary criteria of respiratory exchange ratios in excess of 1.05, and maximal heart rates in excess of their predicted values. Repeatability analyses identified that the typical error (expressed as percent of coefficient of variation) was 20% over 3-months in those reaching volitional exhaustion. CONCLUSION: This work has, for the first time, fully characterised how patients with ILD respond to CPET in terms of primary and secondary verification criteria, and generated novel repeatability data that will prove useful in the assessment of disease progression, and future evaluation of therapeutic regimens where VO2peak is used as an outcome measure.

Feasibility of cardiopulmonary exercise testing in interstitial lung disease: the PETFIB study.

INTRODUCTION: Cardiopulmonary exercise testing (CPET) provides a series of biomarkers, such as peak oxygen uptake, which could assess the development of disease status in interstitial lung disease (ILD). However, despite use in research and clinical settings, the feasibility of CPET in this patient group has yet to be established. METHODS: Twenty-six patients with ILD (19 male) were recruited to this study. Following screening for contraindications to maximal exercise, participants underwent an incremental CPET to volitional exhaustion. Feasibility of CPET was assessed by the implementation, practicality, acceptability and demand, thus providing clinical-driven and patient-driven information on this testing procedure. RESULTS: Of the 26 recruited participants, 24 successfully completed at least one CPET, with 67/78 prospective tests being completed. Contraindications included hypertension, low resting oxygen saturation and recent pulmonary embolism. Of the CPETs undertaken, 63% successfully reached volitional exhaustion, with 31% being terminated early by clinicians due to excessive desaturation. Quantitative and qualitative feedback from participants revealed a positive experience of CPET and desire for it to be included as a future monitoring tool. CONCLUSION: CPET is feasible in patients with ILD. Identification of common clinical contraindications, and understanding of patient perspectives will allow for effective design of future studies utilising CPET as a monitoring procedure.

A Reverse Transcriptase-Cas1 Fusion Protein Contains a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer Acquisition.

Prokaryotic CRISPR-Cas systems provide adaptive immunity by integrating portions of foreign nucleic acids (spacers) into genomic CRISPR arrays. Cas6 proteins then process CRISPR array transcripts into spacer-derived RNAs (CRISPR RNAs; crRNAs) that target Cas nucleases to matching invaders. We find that a Marinomonas mediterranea fusion protein combines three enzymatic domains (Cas6, reverse transcriptase [RT], and Cas1), which function in both crRNA biogenesis and spacer acquisition from RNA and DNA. We report a crystal structure of this divergent Cas6, identify amino acids required for Cas6 activity, show that the Cas6 domain is required for RT activity and RNA spacer acquisition, and demonstrate that CRISPR-repeat binding to Cas6 regulates RT activity. Co-evolution of putative interacting surfaces suggests a specific structural interaction between the Cas6 and RT domains, and phylogenetic analysis reveals repeated, stable association of free-standing Cas6s with CRISPR RTs in multiple microbial lineages, indicating that a functional interaction between these proteins preceded evolution of the fusion.

Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase-Cas1 fusion protein.

CRISPR systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in type I and II CRISPR systems by the acquisition of short segments of DNA (spacers) from invasive elements. In several type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we showed that a RT-Cas1 fusion protein enables the acquisition of RNA spacers in vivo in a RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze the ligation of RNA segments into the CRISPR array, which is followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA.

Loss of miRNAs during processing and storage of cow's (Bos taurus) milk.

MicroRNAs (miRs, miRNAs) play central roles in gene regulation. Previously, we reported that miRNAs from pasteurized, store-bought bovine milk have biological activity in humans. Here, we assessed the effects of milk processing, storage, somatic cell content, and handling by consumers on the degradation of miRNAs in milk; we also quantified miRNAs in dairy products. Pasteurization and homogenization caused a 63% loss of miR-200c, whereas a 67% loss observed for miR-29b was statistically significant only in skim milk. Effects of cold storage and somatic cell content were quantitatively minor (<2% loss). Heating in the microwave caused a 40% loss of miR-29b but no loss of miR-200c. The milk fat content had no effect on miRNA stability during storage and microwave heating. The concentrations of miRNAs in dairy products were considerably lower than in store-bought milk. We conclude that processing of milk by dairies and handling by consumers causes a significant loss of miRNAs.

Feeding and eating disorders in childhood.

OBJECTIVE: To review the literature related to the current DSM-IV-TR diagnostic criteria for feeding disorder of infancy or early childhood; pica; rumination disorder; and other childhood presentations that are characterized by avoidance of food or restricted food intake, with the purpose of informing options for DSM-V. METHOD: Articles were identified by computerized and manual searches and reviewed to evaluate the evidence supporting possible options for revision of criteria. RESULTS: The study of childhood feeding and eating disturbances has been hampered by inconsistencies in classification and use of terminology. Greater clarity around subtypes of feeding and eating problems in children would benefit clinicians and patients alike. DISCUSSION: A number of suggestions supported by existing evidence are made that provide clearer descriptions of subtypes to improve clinical utility and to promote research.