MOLECULAR PHYLOGENETIC ANALYSIS OF GYRINICOLA BATRACHIENSIS (NEMATODA: OXYUROIDEA) LENDS SUPPORT TO THE MONOPHYLY OF THE GENUS AND THE RESURRECTION OF GYRINICOLINAE.

Gyrinicola Yamaguti, 1938, includes 6 species of oxyurid found within the intestinal tract of numerous, larval, anuran species in Europe, Asia, South America, and North America. The systematic placement and hierarchical treatment of the genus has shifted at least 5 times since its discovery; the group was first considered as its own family (Gyrinicolidae), then treated as a subfamily (Gyrinicolinae) of Cosmocercidae, then as a member of the Pharyngodonidae, followed by movement back to the Cosmocercidae, and finally a recent proposal suggested the resurrection of the Gyrinicolidae. Species in the genus vary widely in the morphology of the uterine tract, a characteristic often used to indicate membership in the genus, as it is tied to the reproductive mode. However, until recently very few genetic data were available to aid in the placement of this unique group of worms, and before this study to the best of our knowledge none existed for the North American species. To examine the monophyly and placement of the Gyrinicola we sampled populations of Gyrinicola batrachiensis across North America and screened them for genetic diversity using nuclear markers 18S and 28S. Phylogenies suggest at least 3 clades exist among the nematodes from North America and that these clades, alongside Gyrinicola japonica, form a well-supported group within Oxyuroidea. Further representation of Pharyngodonidae from other vertebrate classes may help clarify the relationship of this historical grouping to other members of the Oxyuroidea.

Raising NAD+ Level Stimulates Short-Chain Dehydrogenase/Reductase Proteins to Alleviate Heart Failure Independent of Mitochondrial Protein Deacetylation.

BACKGROUND: Strategies to increase cellular NAD+ (oxidized nicotinamide adenine dinucleotide) level have prevented cardiac dysfunction in multiple models of heart failure, but molecular mechanisms remain unclear. Little is known about the benefits of NAD+-based therapies in failing hearts after the symptoms of heart failure have appeared. Most pretreatment regimens suggested mechanisms involving activation of sirtuin, especially Sirt3 (sirtuin 3), and mitochondrial protein acetylation. METHODS: We induced cardiac dysfunction by pressure overload in SIRT3-deficient (knockout) mice and compared their response with nicotinamide riboside chloride treatment with wild-type mice. To model a therapeutic approach, we initiated the treatment in mice with established cardiac dysfunction. RESULTS: We found nicotinamide riboside chloride improved mitochondrial function and blunted heart failure progression. Similar benefits were observed in wild-type and knockout mice. Boosting NAD+ level improved the function of NAD(H) redox-sensitive SDR (short-chain dehydrogenase/reductase) family proteins. Upregulation of Mrpp2 (mitochondrial ribonuclease P protein 2), a multifunctional SDR protein and a subunit of mitochondrial ribonuclease P, improves mitochondrial DNA transcripts processing and electron transport chain function. Activation of SDRs in the retinol metabolism pathway stimulates RXRα (retinoid X receptor α)/PPARα (proliferator-activated receptor α) signaling and restores mitochondrial oxidative metabolism. Downregulation of Mrpp2 and impaired mitochondrial ribonuclease P were found in human failing hearts, suggesting a shared mechanism of defective mitochondrial biogenesis in mouse and human heart failure. CONCLUSIONS: These findings identify SDR proteins as important regulators of mitochondrial function and molecular targets of NAD+-based therapy. Furthermore, the benefit is observed regardless of Sirt3-mediated mitochondrial protein deacetylation, a widely held mechanism for NAD+-based therapy for heart failure. The data also show that NAD+-based therapy can be useful in pre-existing heart failure.

Mitochondrial interactome quantitation reveals structural changes in metabolic machinery in the failing murine heart.

Advancements in cross-linking mass spectrometry (XL-MS) bridge the gap between purified systems and native tissue environments, allowing the detection of protein structural interactions in their native state. Here we use isobaric quantitative protein interaction reporter technology (iqPIR) to compare the mitochondria protein interactomes in healthy and hypertrophic murine hearts, 4 weeks post-transaortic constriction. The failing heart interactome includes 588 statistically significant cross-linked peptide pairs altered in the disease condition. We observed an increase in the assembly of ketone oxidation oligomers corresponding to an increase in ketone metabolic utilization; remodeling of NDUA4 interaction in Complex IV, likely contributing to impaired mitochondria respiration; and conformational enrichment of ADP/ATP carrier ADT1, which is non-functional for ADP/ATP translocation but likely possesses non-selective conductivity. Our application of quantitative cross-linking technology in cardiac tissue provides molecular-level insights into the complex mitochondria remodeling in heart failure while bringing forth new hypotheses for pathological mechanisms.

Acetylation of muscle creatine kinase negatively impacts high-energy phosphotransfer in heart failure.

A hallmark of impaired myocardial energetics in failing hearts is the downregulation of the creatine kinase (CK) system. In heart failure patients and animal models, myocardial phosphocreatine content and the flux of the CK reaction are negatively correlated with the outcome of heart failure. While decreased CK activity is highly reproducible in failing hearts, the underlying mechanisms remains elusive. Here, we report an inverse relationship between the activity and acetylation of CK muscle form (CKM) in human and mouse failing hearts. Hyperacetylation of recombinant CKM disrupted MM homodimer formation and reduced enzymatic activity, which could be reversed by sirtuin 2 treatment. Mass spectrometry analysis identified multiple lysine residues on the MM dimer interface, which were hyperacetylated in the failing hearts. Molecular modeling of CK MM homodimer suggested that hyperacetylation prevented dimer formation through interfering salt bridges within and between the 2 monomers. Deacetylation by sirtuin 2 reduced acetylation of the critical lysine residues, improved dimer formation, and restored CKM activity from failing heart tissue. These findings reveal a potentially novel mechanism in the regulation of CK activity and provide a potential target for improving high-energy phosphoryl transfer in heart failure.

NAD(H) in mitochondrial energy transduction: implications for health and disease.

Mitochondria are intracellular organelles that oxidize nutrients, make ATP, and fuel eukaryotic life. Their energy providing function is directly dependent on enzymes and coenzymes contained within the organelle. Perhaps, the most important coenzymes for energy yielding reactions are the pyridine nucleotides NAD(H) and NADP(H). Both aerobic and anaerobic metabolism rely on the electron carrying properties of pyridine nucleotides to regulate energy production. The intracellular NAD+/NADH ratio controls the rate of ATP synthesis by regulating flux through NAD(H)-linked dehydrogenases and by activating NAD+ dependent enzymes that post-translationally modify proteins. Thus, mitochondrial energy transduction pathways can be substantially mediated by NAD+; as an electron carrier exerting control over dehydrogenase enzymes or by activating enzymes that affect protein modification. The importance of this is highlighted in the explosion of recent studies linking impaired NAD+ metabolism to human health and disease. Most notably, studies linking changes in NAD+ availability or altered NAD+/NADH ratio to derangements in metabolic and cellular energy transduction processes. In this review, we focus on the most recent investigative efforts to identify the role NAD+ plays in modulating mitochondrial function and also summarize the current knowledge describing the therapeutic application of elevating NAD+ levels via pharmacologic and genetic approaches to treat human disease.

Audiometric Testing With Pulsed, Steady, and Warble Tones in Listeners With Tinnitus and Hearing Loss.

PURPOSE: This study evaluated the American Speech-Language-Hearing Association's recommendation that audiometric testing for patients with tinnitus should use pulsed or warble tones. Using listeners with varied audiometric configurations and tinnitus statuses, we asked whether steady, pulsed, and warble tones yielded similar audiometric thresholds, and which tone type was preferred. METHOD: Audiometric thresholds (octave frequencies from 0.25-16 kHz) were measured using steady, pulsed, and warble tones in 61 listeners, who were divided into 4 groups on the basis of hearing and tinnitus status. Participants rated the appeal and difficulty of each tone type on a 1-5 scale and selected a preferred type. RESULTS: For all groups, thresholds were lower for warble than for pulsed and steady tones, with the largest effects above 4 kHz. Appeal ratings did not differ across tone type, but the steady tone was rated as more difficult than the warble and pulsed tones. Participants generally preferred pulsed and warble tones. CONCLUSIONS: Pulsed tones provide advantages over steady and warble tones for patients regardless of hearing or tinnitus status. Although listeners preferred pulsed and warble tones to steady tones, pulsed tones are not susceptible to the effects of off-frequency listening, a consideration when testing listeners with sloping audiograms.

Outcomes of decompression for lumbar spinal canal stenosis based upon preoperative radiographic severity.

BACKGROUND: The relationship between severity of preoperative radiographic findings and surgical outcomes following decompression for lumbar degenerative spinal canal stenosis is unclear. Our aim in this paper was to gain insight into this relationship. We determined pre-operative radiographic severity on MRI scans using strict methodological controls and correlated such severity with post-operative outcomes using prospectively collected data. METHODS: Twenty-seven consecutive patients undergoing decompression for isolated degenerative spinal canal stenosis at L4-L5 were included. We measured cross-sectional area on MRI using the technique of Hamanishi. We categorized the severity of stenosis using Laurencin and Lipson's 'Stenosis Ratio'. We determined pre-operative status (prospectively) and post-operative outcomes using Weiner and Fraser's 'Neurogenic Claudication Outcome Score'. We determined patient satisfaction using standardized questionnaires. Each of these is a validated measure. Formal statistical evaluation was undertaken. RESULTS: No patients (0 of 14) with a greater than 50% reduction in cross-sectional area on pre-operative MRI had unsatisfactory outcomes. In contrast, outcomes for patients with less than or equal to 50% reduction in cross-sectional area had unsatifactory outcomes in 6 of 13 cases, with all but one negative outcome having a cross-sectional area reduction between 32% and 47%. CONCLUSION: The findings suggest that there appears to be a relationship between severity of stenosis and outcomes of decompressive surgery such that patients with a greater than 50% reduction in cross sectional area are more likely to have a successful outcome.