Evolution of a biological thermocouple by adaptation of cytochrome c oxidase in a subterrestrial metazoan.

In this study we report a naturally evolved temperature-sensing electrical regulator in the cytochrome c oxidase of the Devil Worm, Halicephalobus mephisto. This extremophile metazoan was isolated 1.3 km underground in a South African goldmine, where it adapted to heat and potentially to hypoxia, making its mitochondrial sequence a likely target of adaptational change. We obtained the full mitochondrial genome sequence of this organism, and show through dN/dS analysis statistically robust evidence of positive selection in H. mephisto cytochrome c oxidase subunits. Seventeen of these positively-selected amino acid substitutions were localized in proximity to the H- and K-pathway proton channels of the complex. Surprisingly, the H. mephisto cytochrome c oxidase proton pump completely shuts down at low temperatures (20°C) leading to approximately a 4.8-fold reduction in the transmembrane proton gradient voltage (ΔΨm) compared to optimal temperature (37°C). Direct measurement of oxygen consumption found a corresponding 4.7-fold drop at 20°C compared to 37°C. Correspondingly, the lifecycle of H. mephisto takes four-fold longer at the low temperature compared to higher. This elegant evolutionary adaptation creates a finely-tuned mitochondrial temperature sensor, allowing this ectothermic organism to maximize its reproductive success in varying environmental temperatures. Our study shows that evolutionary innovation may remodel core metabolism to make it more accurately map onto environmental variation.

The Caenorhabditis elegans TDRD5/7-like protein, LOTR-1, interacts with the helicase ZNFX-1 to balance epigenetic signals in the germline.

LOTUS and Tudor domain containing proteins have critical roles in the germline. Proteins that contain these domains, such as Tejas/Tapas in Drosophila, help localize the Vasa helicase to the germ granules and facilitate piRNA-mediated transposon silencing. The homologous proteins in mammals, TDRD5 and TDRD7, are required during spermiogenesis. Until now, proteins containing both LOTUS and Tudor domains in Caenorhabditis elegans have remained elusive. Here we describe LOTR-1 (D1081.7), which derives its name from its LOTUS and Tudor domains. Interestingly, LOTR-1 docks next to P granules to colocalize with the broadly conserved Z-granule helicase, ZNFX-1. The Tudor domain of LOTR-1 is required for its Z-granule retention. Like znfx-1 mutants, lotr-1 mutants lose small RNAs from the 3' ends of WAGO and mutator targets, reminiscent of the loss of piRNAs from the 3' ends of piRNA precursor transcripts in mouse Tdrd5 mutants. Our work shows that LOTR-1 acts with ZNFX-1 to bring small RNA amplifying mechanisms towards the 3' ends of its RNA templates.

Novel LOTUS-domain proteins are organizational hubs that recruit C. elegans Vasa to germ granules.

We describe MIP-1 and MIP-2, novel paralogous C. elegans germ granule components that interact with the intrinsically disordered MEG-3 protein. These proteins promote P granule condensation, form granules independently of MEG-3 in the postembryonic germ line, and balance each other in regulating P granule growth and localization. MIP-1 and MIP-2 each contain two LOTUS domains and intrinsically disordered regions and form homo- and heterodimers. They bind and anchor the Vasa homolog GLH-1 within P granules and are jointly required for coalescence of MEG-3, GLH-1, and PGL proteins. Animals lacking MIP-1 and MIP-2 show temperature-sensitive embryonic lethality, sterility, and mortal germ lines. Germline phenotypes include defects in stem cell self-renewal, meiotic progression, and gamete differentiation. We propose that these proteins serve as scaffolds and organizing centers for ribonucleoprotein networks within P granules that help recruit and balance essential RNA processing machinery to regulate key developmental transitions in the germ line.

REC-1 and HIM-5 distribute meiotic crossovers and function redundantly in meiotic double-strand break formation in Caenorhabditis elegans.

The Caenorhabditis elegans gene rec-1 was the first genetic locus identified in metazoa to affect the distribution of meiotic crossovers along the chromosome. We report that rec-1 encodes a distant paralog of HIM-5, which was discovered by whole-genome sequencing and confirmed by multiple genome-edited alleles. REC-1 is phosphorylated by cyclin-dependent kinase (CDK) in vitro, and mutation of the CDK consensus sites in REC-1 compromises meiotic crossover distribution in vivo. Unexpectedly, rec-1; him-5 double mutants are synthetic-lethal due to a defect in meiotic double-strand break formation. Thus, we uncovered an unexpected robustness to meiotic DSB formation and crossover positioning that is executed by HIM-5 and REC-1 and regulated by phosphorylation.

CHL-1 provides an essential function affecting cell proliferation and chromosome stability in Caenorhabditis elegans.

A family of helicases that are important in maintaining genome stability is the iron-sulfur group. Members of this family include DOG-1/FANCJ, RTEL1, XPD and Chl1p/DDX11. In Caenorhabitis elegans, the predicted gene M03C11.2 has orthology to the CHL1 (Chromosome loss 1) gene in Saccharomyces cerevisiae and DDX11 (DEAD/H box polypeptide 11) in humans. In this paper, we show that the chl-1 gene in C. elegans is required for normal development and fertility. Mutants have lineage-independent cell proliferation defects that result in a Stu (sterile uncoordinated) phenotype, characterized by gonadal abnormalities and a reduced number of D motor neurons and seam cells. A chromosome stability defect is present in the germ cells, where an abnormal number of DAPI-staining chromosomes appear in diakinesis. CHL-1 function is required for the integrity of poly-guanine/poly-cytosine DNA in the absence of DOG-1/FANCJ: the loss of CHL-1 alone does not result in the deletion of G-tracts, but it does increase the number of deletions observed in the dog-1; chl-1 double mutant, indicating a role for CHL-1 during replication and repair. In addition, we observed that cohesin defects increased the number of deletions in the absence of DOG-1/FANCJ. Our results demonstrate a role for CHL-1 in cell proliferation and maintaining normal chromosome numbers, and implicate CHL-1 in chromosome stability and repair of unresolved secondary structures during replication.

Mixed-valence tetra- and hexanuclear manganese complexes from the flexibility of pyridine-containing beta-diketone ligands.

The reactions of [Mn3O(O2CCCl3)6(H2O)3] with 1-phenyl-3-(2-pyridyl)propane-1,3-dione (HL(1)) and 1-(2-pyridly)-3-(p-tolyl)propane-1,3-dione (HL(2)) in CH2Cl2 afford the mixed-valence Mn(II)2Mn(III)2 tetranuclear complexes [Mn4O(O2CCCl3)6(L(1))2] (1) and [Mn4O(O2CCCl3)6L2(2)] (2), respectively. Similar reactions employing [Mn3O(O2CPh)6(H2O)(py)2] with HL(1) and HL(2) give the Mn(II)3Mn(III)3 hexanuclear complexes [Mn6O2(O2CPh)8(L(1))3] (3) and [Mn6O2(O2CPh)8L3(2)] (4), respectively. Complexes 1.2CH2Cl2, 2.2CH2Cl2.H2O, 3.1.5CH2Cl2.Et2O.H2O, and 4.2CH2Cl2 crystallize in the triclinic space group P1, monoclinic space group P2(1)/c, monoclinic space group P2 1/ n, and monoclinic space group P2(1)/n, respectively. Complexes 1 and 2 consist of a trapped-valence tetranuclear core of [Mn(II)2Mn(III)2(mu4-O)](8+), and complexes 3 and 4 represent a new structural type, possessing a [Mn(II)3Mn(III)3(mu4-O)2](11+) core. The magnetic data indicate that complexes 3 and 4 have a ground-state spin value of S = 7/2 with significant magnetoanisotropy as gauged by the D values of -0.51 cm (-1) and -0.46 cm (-1), respectively, and frequency-dependent out-of-phase signals in alternating current magnetic susceptibility studies indicate their superparamagnetic behavior. In contrast, complexes 1 and 2 are low-spin molecules with an S = 1 ground state. Single-molecule magnetism behavior confirmed for 3 the presence of sweep-rate and temperature-dependent hysteresis loops in single-crystal M versus H studies at temperatures down to 40 mK.

Myocardial perfusion assessment in patients with medium probability of coronary artery disease and no prior myocardial infarction: comparison of myocardial contrast echocardiography with 99mTc single-photon emission computed tomography.

BACKGROUND: No previous studies have compared myocardial contrast echocardiography (MCE) and single-photon emission computerized tomography (SPECT) for the detection of coronary artery disease (CAD) in patients with a medium likelihood of CAD. This study was developed to test the hypothesis that MCE is superior to SPECT for the detection of CAD. METHODS: Fifty-five patients with a medium probability of CAD and no previous myocardial infarction were recruited. Results of MCE and SPECT were compared to quantitative coronary arteriography, where CAD was defined as >50% stenosis. Each patient was examined for the anterior and posterior circulations. RESULTS: On a coronary circulation basis (n = 110), the sensitivity of MCE was significantly greater than that of SPECT for the detection of CAD (86% vs 43%, P <.0001). However, the specificities were similar (88% and 93%, P =.52). Both techniques were marginally more accurate in the anterior compared to the posterior circulation (88% vs 76%, P =.07 for MCE and 79% vs 63%, P =.19, for SPECT). On a patient basis (n = 55), MCE had a higher sensitivity than SPECT for the detection of CAD (83% vs 49%, P <.05). Although specificity tended to be higher for SPECT than MCE (92% vs 58%), it was not significant (P =.33). When CAD was defined as >40% coronary stenosis, the specificity of MCE increased to 83% without any change in sensitivity. CONCLUSION: In this preliminary study, MCE was found to be superior to SPECT during dipyridamole stress for the diagnosis of CAD in patients with a medium pretest probability of CAD. Larger studies are required to confirm these findings.

Prognostic value of normal stress echocardiogram in patients with suspected coronary artery disease--a British general hospital experience.

AIMS: To determine the prognostic value of a normal stress echocardiogram in the setting of a large district general non-university hospital in the United Kingdom. METHODS: Between January 1996 and December 1999, all patients who had undergone stress echocardiography were identified and those with normal results were studied. Normal stress echocardiograms were found in 252 patients, 19 of whom were lost to follow-up. Deaths and nonfatal myocardial infarctions were considered hard cardiac events and data was collected in the remaining 233 patients. RESULTS: Among the 233 patients, the pre-test probability of coronary artery disease was low in 68 (27.9%) and intermediate or high in 168 (72.1%). During a follow-up period of mean (SD) 2.7 (1.1) years, death occurred in 4 patients of which 3 were consequent to acute myocardial infarctions and 1 was unexplained. One patient sustained a nonfatal infarction. Thus, the annualized mortality and hard event rates were 0.6% per patient/year and 0.8% per patient/year, respectively. CONCLUSIONS: A normal stress echocardiogram portends an excellent prognosis, even in a cohort with a high proportion of patients having intermediate or high pre-test probability of coronary artery disease.