Transcript availability dictates the balance between strand-asynchronous and strand-coupled mitochondrial DNA replication.

Mammalian mitochondria operate multiple mechanisms of DNA replication. In many cells and tissues a strand-asynchronous mechanism predominates over coupled leading and lagging-strand DNA synthesis. However, little is known of the factors that control or influence the different mechanisms of replication, and the idea that strand-asynchronous replication entails transient incorporation of transcripts (aka bootlaces) is controversial. A firm prediction of the bootlace model is that it depends on mitochondrial transcripts. Here, we show that elevated expression of Twinkle DNA helicase in human mitochondria induces bidirectional, coupled leading and lagging-strand DNA synthesis, at the expense of strand-asynchronous replication; and this switch is accompanied by decreases in the steady-state level of some mitochondrial transcripts. However, in the so-called minor arc of mitochondrial DNA where transcript levels remain high, the strand-asynchronous replication mechanism is instated. Hence, replication switches to a strand-coupled mechanism only where transcripts are scarce, thereby establishing a direct correlation between transcript availability and the mechanism of replication. Thus, these findings support a critical role of mitochondrial transcripts in the strand-asynchronous mechanism of mitochondrial DNA replication; and, as a corollary, mitochondrial RNA availability and RNA/DNA hybrid formation offer means of regulating the mechanisms of DNA replication in the organelle.

Beyond Messiaen's birds: the post-verbal world of dementia.

This paper investigates the use of verbatim musical transcription as a research method in dementia care. It reports on an art-based ethnographic study (Aesthetic Research in Everyday Life (Aeriel)) in which verbatim transcription was applied to everyday interactions in dementia care, making use of musical-instead of verbal-notation. Starting from the notion that medical and healthcare settings can be sites of 'found performance', the paper reviews literature relating to artistic methodologies within medical humanities, music, ethnography and dementia care. From this review, it proposes a research design and method of verbatim musical transcription as a potential avenue of investigating communication between carer and cared for in dementia care. The paper offers an illustrative example from Aeriel and draws conclusions from the synthesis of verbal and musical data analysis. Findings indicate an important advance in studies of dementia care communication towards a concept of the 'post-verbal' enabled by a musical research method and the clinical applications that it offers.

Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication.

Encoding ribonuclease H1 (RNase H1) degrades RNA hybridized to DNA, and its function is essential for mitochondrial DNA maintenance in the developing mouse. Here we define the role of RNase H1 in mitochondrial DNA replication. Analysis of replicating mitochondrial DNA in embryonic fibroblasts lacking RNase H1 reveals retention of three primers in the major noncoding region (NCR) and one at the prominent lagging-strand initiation site termed Ori-L. Primer retention does not lead immediately to depletion, as the persistent RNA is fully incorporated in mitochondrial DNA. However, the retained primers present an obstacle to the mitochondrial DNA polymerase γ in subsequent rounds of replication and lead to the catastrophic generation of a double-strand break at the origin when the resulting gapped molecules are copied. Hence, the essential role of RNase H1 in mitochondrial DNA replication is the removal of primers at the origin of replication.

Disposition, metabolism and mass balance of delafloxacin in healthy human volunteers following intravenous administration.

1. The pharmacokinetics and disposition of delafloxacin was investigated following a single intravenous (300 mg, 100 µCi) dose to healthy male subjects. 2. Mean Cmax, AUC0-∞, Tmax and t1/2 values for delafloxacin were 8.98 µg/mL, 21.31 µg h/mL, 1 h and 2.35 h, respectively, after intravenous dosing. 3. Radioactivity was predominantly excreted via the kidney with 66% of the radioactive dose recovered in the urine. Approximately 29% of the radioactivity was recovered in the faeces, giving an overall mean recovery of 94% administered radioactivity. 4. The predominant circulating components were identified as delafloxacin and a direct glucuronide conjugate of delafloxacin.

Mitochondrial DNA replication proceeds via a 'bootlace' mechanism involving the incorporation of processed transcripts.

The observation that long tracts of RNA are associated with replicating molecules of mitochondrial DNA (mtDNA) suggests that the mitochondrial genome of mammals is copied by an unorthodox mechanism. Here we show that these RNA-containing species are present in living cells and tissue, based on interstrand cross-linking. Using DNA synthesis in organello, we demonstrate that isolated mitochondria incorporate radiolabeled RNA precursors, as well as DNA precursors, into replicating DNA molecules. RNA-containing replication intermediates are chased into mature mtDNA, to which they are thus in precursor-product relationship. While a DNA chain terminator rapidly blocks the labeling of mitochondrial replication intermediates, an RNA chain terminator does not. Furthermore, processed L-strand transcripts can be recovered from gel-extracted mtDNA replication intermediates. Therefore, instead of concurrent DNA and RNA synthesis, respectively, on the leading and lagging strands, preformed processed RNA is incorporated as a provisional lagging strand during mtDNA replication. These findings indicate that RITOLS is a physiological mechanism of mtDNA replication, and that it involves a 'bootlace' mechanism, in which processed transcripts are successively hybridized to the lagging-strand template, as the replication fork advances.

Alternative translation initiation augments the human mitochondrial proteome.

Alternative translation initiation (ATI) is a mechanism of producing multiple proteins from a single transcript, which in some cases regulates trafficking of proteins to different cellular compartments, including mitochondria. Application of a genome-wide computational screen predicts a cryptic mitochondrial targeting signal for 126 proteins in mouse and man that is revealed when an AUG codon located downstream from the canonical initiator methionine codon is used as a translation start site, which we term downstream ATI (dATI). Experimental evidence in support of dATI is provided by immunoblotting of endogenous truncated proteins enriched in mitochondrial cell fractions or of co-localization with mitochondria using immunocytochemistry. More detailed cellular localization studies establish mitochondrial targeting of a member of the cytosolic poly(A) binding protein family, PABPC5, and of the RNA/DNA helicase PIF1α. The mitochondrial isoform of PABPC5 co-immunoprecipitates with the mitochondrial poly(A) polymerase, and is markedly reduced in abundance when mitochondrial DNA and RNA are depleted, suggesting it plays a role in RNA metabolism in the organelle. Like PABPC5 and PIF1α, most of the candidates identified by the screen are not currently annotated as mitochondrial proteins, and so dATI expands the human mitochondrial proteome.

Discordance of the Urinary and Pleural Fluid Antigen Test and False Positive for Streptococcus pneumoniae in Empyema Secondary to Necrotizing Bacterial Pneumonia.

Empyema is a severe complication of pneumonia with high morbidity and mortality rates. Rapid diagnosis and tailoring of antibiotic therapy are crucial to treatment success for these severe bacterial lung infections. A Streptococcus pneumoniae (S. pneumonia) antigen test drawn from the pleural fluid rather than a urine sample has been found to have equivalent diagnostic utility to the urinary antigen test. Discordance between these tests is rare. We report a case of a 69-year-old female with CT imaging findings consistent with empyema and a bronchopulmonary fistula. A rapid S. pneumonia antigen test was negative from the urinary sample but positive when drawn from a patient's pleural fluid sample. Final pleural fluid cultures resulted in Streptococcus constellatus (S. constellatus). This case demonstrates discordance between the results of urinary and pleural fluid S. pneumoniae antigen tests, representing a potential pitfall in using rapid antigen testing on pleural fluid samples. False positives for the S. pneumoniae antigen in patients with viridans streptococci infections have been documented due to the cross-reactivity of cell wall proteins in different streptococcal species. Physicians encountering bacterial pneumonia of unknown etiology complicated by empyema should understand the potential for discordance and false positives using this diagnostic method.

The Curious Case of Elevated Tryptase: Workup and Differential in Family of Four.

Elevated basal serum tryptase (BST) levels are markers of both mast cell activation and overall mast cell burden. We present a family of four individuals with elevated tryptase levels greater than or equal to 20 mcg/L, all of whom exhibited signs and symptoms suggestive of mast cell activation. Differential diagnoses included hereditary alpha tryptasemia (HaT), systemic mastocytosis (SM), and mast cell activation syndrome (MCAS). In three individuals, SM was ruled out with normal morphology on bone marrow biopsy combined with negative genetic markers. Further workup would be required for the diagnosis of MCAS since serum tryptase levels were not obtained in our emergency department during acute episodes. Although genetic testing for HaT was not available upon initial workup, HaT remains the most likely explanation for this family's elevated BST.

A Case of Lyme Carditis With Variable Heart Block Successfully Treated With Oral Doxycycline.

A 39-year-old male without significant past medical history presented with three weeks of worsening fatigue, migratory arthralgia, rash, and unilateral facial weakness after spending three months in Vermont. Serology showed positive Lyme titers 1:64 for both IgM and IgG. EKG on presentation showed a P-R interval of 384 ms, and the patient was admitted for concern of Lyme carditis. Serial EKGs obtained throughout his stay demonstrated variability between first- and second-degree heart blocks. After consultation with Infectious Disease, he was transitioned to oral doxycycline to complete a 21-day course. The patient's heart block and other symptoms had resolved on follow-up after the treatment course had been completed.

Low-Virulence Organisms and Periprosthetic Joint Infection-Biofilm Considerations of These Organisms.

PURPOSE OF REVIEW: The purpose of this manuscript is to provide a critical review of peer-reviewed literature over the last 5 years related to low virulent organisms associated with periprosthetic joint infection (PJI). We evaluated the most common organisms, the diagnostic challenges, and the novel tools available in the perioperative workup of PJI as well as the current understanding of how biofilm potentiates the indolent clinical presentation and explore a possible shift in the surgical management of these patients. RECENT FINDINGS: Biofilm actively prevents macrophage phagocytosis by suppressing proinflammatory activity through the recruitment of myeloid-derived suppressor cells. Given the appropriate host and organism conditions, increased utilization of one-stage exchange arthroplasty in the surgical treatment of these low virulent infections may be on the rise. Biomarkers and molecular techniques offer encouraging results to diagnose low virulent organisms and future research focused on the disruption of biofilm may ultimately give rise to improved treatment strategies.

Influenza-Like Illness in Travelers to the Developing World.

Travelers to developing regions are at risk for development of influenza-like illness (ILI). Little is known of traveler and trip characteristics associated with the development of ILI. TravMil is a prospective observational study, enrolling subjects presenting to six military travel clinics or predeployment-screening sites. We analyzed pre- and post-travel surveys from travelers visiting regions outside of the continental United States, Western or Northern Europe, Canada, Australia, or New Zealand between January 2010 and March 2016. Influenza-like illness was defined as a self-reported fever associated with either sore throat or cough. Trip and traveler characteristics were analyzed to determine risk factors for the development of ILI. Two thousand nine hundred and thirty-two trips were recorded (55% male, median age 45 years, 69% white, 51% on vacation, median travel duration 17 days). The 2,337 trips included the number of self-reported influenza vaccinations in the preceding 5 years (median 5). Eleven percent of the trips were complicated by an ILI lasting a median of 5 days; 70% and 17% of these reported upper and lower respiratory tract infection, respectively, and 12% reported both. On multivariate analysis, increased risk of ILI was associated with female gender (odds ratio [OR]: 1.60 [confidence interval (CI): 1.25-2.05], P < 0.01), age (years) (OR: 1.01 [CI: 1.01-1.02], P < 0.01); and duration of travel (days) (OR: 1.01 [CI: 1.00-1.01], P < 0.01). Influenza-like illness is common in travelers, regardless of traveler characteristics, purpose of travel, destination, or season of year. Female gender, older age, and longer duration of travel were associated with an increased risk of ILI. Additional tools and strategies are needed to prevent ILI in international travelers.

"Found Performance": Towards a Musical Methodology for Exploring the Aesthetics of Care.

Concepts of performance in fine art reflect key processes in music therapy. Music therapy enables practitioners to reframe patients as performers, producing new meanings around the clinical knowledge attached to medical histories and constructs. In this paper, music therapy practices are considered in the wider context of art history, with reference to allied theories from social research. Tracing a century in art that has revised the performativity of found objects (starting with Duchamp's "Fountain"), and of found sound (crystallised by Cage's 4' 33) this paper proposes that music therapy might be a pioneer methodology of "found performance". Examples from music therapy and contemporary socially engaged art practices are brought as potential links between artistic methodologies and medical humanities research, with specific reference to notions of Aesthetics of Care.

Characterizing the mitochondrial DNA polymerase gamma interactome by BioID identifies Ruvbl2 localizes to the mitochondria.

Human mitochondrial DNA (mtDNA) is replicated by the mitochondrial DNA polymerase gamma (POLG). Using proximity dependent biotin labelling (BioID), we characterized the POLG interactome and identified new interaction partners involved in mtDNA maintenance, transcription, translation and protein quality control. We also identified interaction with the nuclear AAA+ ATPase Ruvbl2, suggesting mitochondrial localization for this protein. Ruvbl2 was detected in mitochondria-enriched fractions in leukemic cells. Additionally, transgenic overexpression of Ruvbl2 from an alternative translation initiation site resulted in mitochondrial co-localization. Overall, POLG interactome mapping identifies novel proteins which support mitochondrial biogenesis and a potential novel mitochondrial isoform of Ruvbl2.

Analysis of Replicating Mitochondrial DNA by In Organello Labeling and Two-Dimensional Agarose Gel Electrophoresis.

Our understanding of the mechanisms of DNA replication in a broad range of organisms and viruses has benefited from the application of two-dimensional agarose gel electrophoresis (2D-AGE). The method resolves DNA molecules on the basis of size and shape and is technically straightforward. 2D-AGE sparked controversy in the field of mitochondria when it revealed replicating molecules with lengthy tracts of RNA, a phenomenon never before reported in nature. More recently, radioisotope labeling of the DNA in the mitochondria has been coupled with 2D-AGE. In its first application, this procedure helped to delineate the "bootlace mechanism of mitochondrial DNA replication," in which processed mitochondrial transcripts are hybridized to the lagging strand template at the replication fork as the leading DNA strand is synthesized. This chapter provides details of the method, how it has been applied to date and concludes with some potential future applications of the technique.

The 'ripple effect': Towards researching improvisational music therapy in dementia care homes.

Increased interest in, and demand for, music therapy provision for persons with dementia prompted this study's exploration of music therapists' strategies for creating musical communities in dementia care settings, considering the needs and resources of people affected by dementia. Focus group discussions and detailed iterative study of improvisational music therapy work by six experienced practitioners clarify the contextual immediacy and socio-musical complexities of music therapy in dementia care homes. Music therapy's 'ripple effect', with resonances from micro (person-to-person musicking), to meso (musicking beyond 'session time') and macro level (within the care home and beyond), implies that all who are part of the dementia care ecology need opportunities for flourishing, shared participation, and for expanded self-identities; beyond 'staff', 'residents', or 'being in distress'. On such basis, managers and funders might consider an extended brief for music therapists' roles, to include generating and maintaining musical wellbeing throughout residential care settings.

Quantitative whole-body autoradiography, LC-MS/MS and MALDI for drug-distribution studies in biological samples: the ultimate matrix trilogy.

The drug-development process requires an understanding of the ADME properties of the novel therapeutic agent. Determination of drug concentrations and identity in excreta (urine and feces) examines the products of these processes. Similar measurements made on plasma, while accurately determining exposure, show only what is being transported around the body. Both activities fail to confirm the nature of components at the pharmacologically relevant matrix - the tissue. Attention is therefore being directed towards methods that can be employed to address this lack in our current methodologies, to provide better quality data on which risk assessments can be made, so that pharmacological models can be refined, and drug safety improved. In this article, we will look at the current methods used to obtain tissue drug and drug metabolite concentrations, and their potential use in drug discovery.

A cryptic targeting signal creates a mitochondrial FEN1 isoform with tailed R-Loop binding properties.

A growing number of DNA transacting proteins is found in the nucleus and in mitochondria, including the DNA repair and replication protein Flap endonuclease 1, FEN1. Here we show a truncated FEN1 isoform is generated by alternative translation initiation, exposing a mitochondrial targeting signal. The shortened form of FEN1, which we term FENMIT, localizes to mitochondria, based on import into isolated organelles, immunocytochemistry and subcellular fractionation. In vitro FENMIT binds to flap structures containing a 5' RNA flap, and prefers such substrates to single-stranded RNA. FENMIT can also bind to R-loops, and to a lesser extent to D-loops. Exposing human cells to ethidium bromide results in the generation of RNA/DNA hybrids near the origin of mitochondrial DNA replication. FENMIT is recruited to the DNA under these conditions, and is released by RNase treatment. Moreover, high levels of recombinant FENMIT expression inhibit mtDNA replication, following ethidium bromide treatment. These findings suggest FENMIT interacts with RNA/DNA hybrids in mitochondrial DNA, such as those found at the origin of replication.

Mammalian mitochondrial DNA replication intermediates are essentially duplex but contain extensive tracts of RNA/DNA hybrid.

We demonstrate, using transmission electron microscopy and immunopurification with an antibody specific for RNA/DNA hybrid, that intact mitochondrial DNA replication intermediates are essentially duplex throughout their length but contain extensive RNA tracts on one strand. However, the extent of preservation of RNA in such molecules is highly dependent on the preparative method used. These findings strongly support the strand-coupled model of mitochondrial DNA replication involving RNA incorporation throughout the lagging strand.

A soluble RecN homologue provides means for biochemical and genetic analysis of DNA double-strand break repair in Escherichia coli.

RecN is a highly conserved, SMC-like protein in bacteria. It plays an important role in the repair of DNA double-strand breaks and is therefore a key factor in maintaining genome integrity. The insolubility of Escherichia coli RecN has limited efforts to unravel its function. We overcame this limitation by replacing the resident coding sequence with that of Haemophilus influenzae RecN. The heterologous construct expresses Haemophilus RecN from the SOS-inducible E. coli promoter. The hybrid gene is fully functional, promoting survival after I-SceI induced DNA breakage, gamma irradiation or exposure to mitomycin C as effectively as the native gene, indicating that the repair activity is conserved between these two species. H. influenzae RecN is quite soluble, even when expressed at high levels, and is readily purified. Its analysis by ionisation-mass spectrometry, gel filtration and glutaraldehyde crosslinking indicates that it is probably a dimer under physiological conditions, although a higher multimer cannot be excluded. The purified protein displays a weak ATPase activity that is essential for its DNA repair function in vivo. However, no DNA-binding activity was detected, which contrasts with RecN from Bacillus subtilis. RecN proteins from Aquifex aeolicus and Bacteriodes fragilis also proved soluble. Neither binds DNA, but the Aquifex RecN has weak ATPase activity. Our findings support studies indicating that RecN, and the SOS response in general, behave differently in E. coli and B. subtilis. The hybrid recN reported provides new opportunities to study the genetics and biochemistry of how RecN operates in E. coli.