Dealing with an Unconventional Genetic Code in Mitochondria: The Biogenesis and Pathogenic Defects of the 5‐Formylcytosine Modification in Mitochondrial tRNAMet^{Met}

Human mitochondria contain their own genome, which uses an unconventional genetic code. In addition to the standard AUG methionine codon, the single mitochondrial tRNA Methionine (mt‐tRNA$^{Met}$) also recognises AUA during translation initiation and elongation. Post‐transcriptional modifications of tRNAs are important for structure, stability, correct folding and aminoacylation as well as decoding. The unique 5‐formylcytosine (f$^{5}$C) modification of position 34 in mt‐tRNA$^{Met}$ has been long postulated to be crucial for decoding of unconventional methionine codons and efficient mitochondrial translation. However, the enzymes responsible for the formation of mitochondrial f$^{5}$C have been identified only recently. The first step of the f$^{5}$C pathway consists of methylation of cytosine by NSUN3. This is followed by further oxidation by ABH1. Here, we review the role of f$^{5}$C, the latest breakthroughs in our understanding of the biogenesis of this unique mitochondrial tRNA modification and its involvement in human disease.Medical Research Council, UK is gratefully acknowledged for generous support of this work

Concentration and localization of zinc during seed development and germination in wheat

In a field experiment, the effect of foliar Zn applications on the concentration of Zn in seeds of a bread wheat cultivar (Triticum aestivum L. cv. Balatilla) was studied during different stages of seed development. In addition, a staining method using dithizone (DTZ: diphenyl thiocarbazone) was applied to (1) study the localization of Zn in seeds, (2) follow the remobilization of Zn during germination, and (3) develop a rapid visual Zn screening method for seed and flour samples. In all seed development stages, foliar Zn treatments were effective in increasing seed Zn concentration. The highest Zn concentration in the seeds was found in the first stage of seed development (around the early milk stage); after this, seed Zn concentration gradually decreased until maturity. When reacting with Zn, DTZ forms a redcolored complex. The DTZ staining of seed samples revealed that Zn is predominantly located in the embryo and aleurone parts of the seeds. After 36 h of germination, the coleoptile and roots that emerged from seeds showed very intensive red color formation and had Zn concentrations up to 200 mg kg1, indicating a substantial remobilization of Zn from seed pools into the developing roots (radicle) and coleoptile. The DTZ staining method seems to be useful in ranking flour samples for their Zn concentrations. There was a close relationship between the seed Zn concentrations and spectral absorbance of the methanol extracts of the flour samples stained with DTZ. The results suggest that (1) accumulation of Zn in seeds is particularly high during early seed development, (2) Zn is concentrated in the embryo and aleurone parts, and (3) the DTZ staining method can be used as a rapid, semiquantitative method to estimate Zn concentrations of flour and seed samples and to screen genotypes for their Zn concentrations in seeds

A field trial of a reusable, hollow, cast-in-situ pile

This paper describes the concept and field testing of a hollow, cast-in-situ, rotary bored pile foundation 1200 mm diameter and 30 m deep. The aim of the foundation is to allow large-diameter piles to be constructed using less concrete than in an equivalent conventional solid pile, and with a view to allowing reuse at a later date. Reuse is made possible because the hollow core of the pile allows access for inspection after demolition of an existing structure. The new piles may also allow modification to enhance load capacity by augering through the base and extending their length. In addition, the piles are better suited than conventional piles for use as ‘energy piles' to allow environmentally friendly heating and cooling. The geotechnical performance of the hollow test pile was comparable with that of a conventional solid pile constructed during the same trial. Details of construction are given, including lessons learned

New directions in cellular therapy of cancer: a summary of the summit on cellular therapy for cancer

A summit on cellular therapy for cancer discussed and presented advances related to the use of adoptive cellular therapy for melanoma and other cancers. The summit revealed that this field is advancing rapidly. Conventional cellular therapies, such as tumor infiltrating lymphocytes (TIL), are becoming more effective and more available. Gene therapy is becoming an important tool in adoptive cell therapy. Lymphocytes are being engineered to express high affinity T cell receptors (TCRs), chimeric antibody-T cell receptors (CARs) and cytokines. T cell subsets with more naïve and stem cell-like characteristics have been shown in pre-clinical models to be more effective than unselected populations and it is now possible to reprogram T cells and to produce T cells with stem cell characteristics. In the future, combinations of adoptive transfer of T cells and specific vaccination against the cognate antigen can be envisaged to further enhance the effectiveness of these therapies

Tips and Traps: Lessons From Codesigning a Clinician E-Monitoring Tool for Computerized Cognitive Behavioral Therapy

Background: Computerized cognitive behavioral therapy (cCBT) is an acceptable and promising treatment modality for adolescents with mild-to-moderate depression. Many cCBT programs are standalone packages with no way for clinicians to monitor progress or outcomes. We sought to develop an electronic monitoring (e-monitoring) tool in consultation with clinicians and adolescents to allow clinicians to monitor mood, risk, and treatment adherence of adolescents completing a cCBT program called SPARX (Smart, Positive, Active, Realistic, X-factor thoughts). Objective: The objectives of our study were as follows: (1) assess clinicians’ and adolescents’ views on using an e-monitoring tool and to use this information to help shape the development of the tool and (2) assess clinician experiences with a fully developed version of the tool that was implemented in their clinical service. Methods: A descriptive qualitative study using semi-structured focus groups was conducted in New Zealand. In total, 7 focus groups included clinicians (n=50) who worked in primary care, and 3 separate groups included adolescents (n=29). Clinicians were general practitioners (GPs), school guidance counselors, clinical psychologists, youth workers, and nurses. Adolescents were recruited from health services and a high school. Focus groups were run to enable feedback at 3 phases that corresponded to the consultation, development, and post-implementation stages. Thematic analysis was applied to transcribed responses. Results: Focus groups during the consultation and development phases revealed the need for a simple e-monitoring registration process with guides for end users. Common concerns were raised in relation to clinical burden, monitoring risk (and effects on the therapeutic relationship), alongside confidentiality or privacy and technical considerations. Adolescents did not want to use their social media login credentials for e-monitoring, as they valued their privacy. However, adolescents did want information on seeking help, and personalized monitoring and communication arrangements. Post-implementation, clinicians who had used the tool in practice revealed no adverse impact on the therapeutic relationship, and adolescents were not concerned about being e-monitored. Clinicians did need additional time to monitor adolescents, and the e-monitoring tool was used in a different way than was originally anticipated. Also, it was suggested that the registration process could be further streamlined and integrated with existing clinical data management systems, and the use of clinician alerts could be expanded beyond the scope of simply flagging adolescents of concern. Conclusions: An e-monitoring tool was developed in consultation with clinicians and adolescents. However, the study revealed the complexity of implementing the tool in clinical practice. Of salience were privacy, parallel monitoring systems, integration with existing electronic medical record systems, customization of the e-monitor, and pre-agreed monitoring arrangements between clinicians and adolescents

TRNT1 deficiency: clinical, biochemical and molecular genetic features

BACKGROUND: TRNT1 (CCA-adding transfer RNA nucleotidyl transferase) enzyme deficiency is a new metabolic disease caused by defective post-transcriptional modification of mitochondrial and cytosolic transfer RNAs (tRNAs). RESULTS: We investigated four patients from two families with infantile-onset cyclical, aseptic febrile episodes with vomiting and diarrhoea, global electrolyte imbalance during these episodes, sideroblastic anaemia, B lymphocyte immunodeficiency, retinitis pigmentosa, hepatosplenomegaly, exocrine pancreatic insufficiency and renal tubulopathy. Other clinical features found in children include sensorineural deafness, cerebellar atrophy, brittle hair, partial villous atrophy and nephrocalcinosis. Whole exome sequencing and bioinformatic filtering were utilised to identify recessive compound heterozygous TRNT1 mutations (missense mutation c.668T>C, p.Ile223Thr and a novel splice mutation c.342+5G>T) segregating with disease in the first family. The second family was found to have a homozygous TRNT1 mutation (c.569G>T), p.Arg190Ile, (previously published). We found normal mitochondrial translation products using passage matched controls and functional perturbation of 3' CCA addition to mitochondrial tRNAs (tRNA(Cys), tRNA(LeuUUR) and tRNA(His)) in fibroblasts from two patients, demonstrating a pathomechanism affecting the CCA addition to mt-tRNAs. Acute management of these patients included transfusion for anaemia, fluid and electrolyte replacement and immunoglobulin therapy. We also describe three-year follow-up findings after treatment by bone marrow transplantation in one patient, with resolution of fever and reversal of the abnormal metabolic profile. CONCLUSIONS: Our report highlights that TRNT1 mutations cause a spectrum of disease ranging from a childhood-onset complex disease with manifestations in most organs to an adult-onset isolated retinitis pigmentosa presentation. Systematic review of all TRNT1 cases and mutations reported to date revealed a distinctive phenotypic spectrum and metabolic and other investigative findings, which will facilitate rapid clinical recognition of future cases

The pestivirus N terminal protease N(pro) redistributes to mitochondria and peroxisomes suggesting new sites for regulation of IRF3 by N(pro.)

The N-terminal protease of pestiviruses, N(pro) is a unique viral protein, both because it is a distinct autoprotease that cleaves itself from the following polyprotein chain, and also because it binds and inactivates IRF3, a central regulator of interferon production. An important question remains the role of N(pro) in the inhibition of apoptosis. In this study, apoptotic signals induced by staurosporine, interferon, double stranded RNA, sodium arsenate and hydrogen peroxide were inhibited by expression of wild type N(pro), but not by mutant protein N(pro) C112R, which we show is less efficient at promoting degradation of IRF3, and led to the conclusion that N(pro) inhibits the stress-induced intrinsic mitochondrial pathway through inhibition of IRF3-dependent Bax activation. Both expression of N(pro) and infection with Bovine Viral Diarrhea Virus (BVDV) prevented Bax redistribution and mitochondrial fragmentation. Given the role played by signaling platforms during IRF3 activation, we have studied the subcellular distribution of N(pro) and we show that, in common with many other viral proteins, N(pro) targets mitochondria to inhibit apoptosis in response to cell stress. N(pro) itself not only relocated to mitochondria but in addition, both N(pro) and IRF3 associated with peroxisomes, with over 85% of N(pro) puncta co-distributing with PMP70, a marker for peroxisomes. In addition, peroxisomes containing N(pro) and IRF3 associated with ubiquitin. IRF3 was degraded, whereas N(pro) accumulated in response to cell stress. These results implicate mitochondria and peroxisomes as new sites for IRF3 regulation by N(pro), and highlight the role of these organelles in the anti-viral pathway

Receptor protein tyrosine phosphatases are novel components of the polycystin complex

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutation of PKD1 and PKD2 that encode polycystin-1 and polycystin-2. Polycystin-1 is tyrosine phosphorylated and modulates multiple signaling pathways including AP-1, and the identity of the phosphatases regulating polycystin-1 are previously uncharacterized. Here we identify members of the LAR protein tyrosine phosphatase (RPTP) superfamily as members of the polycystin-1complex mediated through extra- and intracellular interactions. The first extracellular PKD1 domain of polycystin-1 interacts with the first Ig domain of RPTPσ, while the polycystin-1 C-terminus of polycystin-1 interacts with the regulatory D2 phosphatase domain of RPTPγ. Additional homo- and heterotypic interactions between RPTPs recruit RPTPδ. The multimeric polycystin protein complex is found localised in cilia. RPTPσ and RPTPδ are also part of a polycystin-1/E-cadherin complex known to be important for early events in adherens junction stabilisation. The interaction between polycystin-1 and RPTPγ is disrupted in ADPKD cells, while RPTPσ and RPTPδ remain closely associated with E-cadherin, largely in an intracellular location. The polycystin-1 C-terminus is an in vitro substrate of RPTPγ, which dephosphorylates the c-Src phosphorylated Y4237 residue and activates AP1-mediated transcription. The data identify RPTPs as novel interacting partners of the polycystins both in cilia and at adhesion complexes and demonstrate RPTPγ phosphatase activity is central to the molecular mechanisms governing polycystin-dependent signaling. This article is part of a Special Issue entitled: Polycystic Kidney Disease