The Killing of African Trypanosomes by Ethidium Bromide

Introduced in the 1950s, ethidium bromide (EB) is still used as an anti-trypanosomal drug for African cattle although its mechanism of killing has been unclear and controversial. EB has long been known to cause loss of the mitochondrial genome, named kinetoplast DNA (kDNA), a giant network of interlocked minicircles and maxicircles. However, the existence of viable parasites lacking kDNA (dyskinetoplastic) led many to think that kDNA loss could not be the mechanism of killing. When recent studies indicated that kDNA is indeed essential in bloodstream trypanosomes and that dyskinetoplastic cells survive only if they have a compensating mutation in the nuclear genome, we investigated the effect of EB on kDNA and its replication. We here report some remarkable effects of EB. Using EM and other techniques, we found that binding of EB to network minicircles is low, probably because of their association with proteins that prevent helix unwinding. In contrast, covalently-closed minicircles that had been released from the network for replication bind EB extensively, causing them, after isolation, to become highly supertwisted and to develop regions of left-handed Z-DNA (without EB, these circles are fully relaxed). In vivo, EB causes helix distortion of free minicircles, preventing replication initiation and resulting in kDNA loss and cell death. Unexpectedly, EB also kills dyskinetoplastic trypanosomes, lacking kDNA, by inhibiting nuclear replication. Since the effect on kDNA occurs at a >10-fold lower EB concentration than that on nuclear DNA, we conclude that minicircle replication initiation is likely EB's most vulnerable target, but the effect on nuclear replication may also contribute to cell killing

A New Method to Constrain Neutron Star Structure from Quasi-periodic Oscillations

We develop a new method to measure neutron star (NS) parameters and derive constraints on the equation of state (EoS) of dense matter by fitting the frequencies of simultaneous quasi-periodic oscillation (QPO) modes observed in the X-ray flux of accreting NSs in low-mass X-ray binaries. To this aim, we calculate the fundamental frequencies of geodesic motion around rotating NSs based on an accurate general-relativistic approximation for their external spacetime. Once the fundamental frequencies are related to the observed frequencies through a QPO model, they can be fit to the data to obtain estimates of the three parameters describing the spacetime, namely the NS mass, angular momentum and quadrupole moment. From these parameters we derive information on the NS structure and EoS. We present a proof of principle of our method applied to pairs of kHz QPO frequencies observed from three systems (4U1608-52, 4U0614+09, and 4U1728-34). We identify the kHz QPOs with the azimuthal and the periastron precession frequencies of matter orbiting the NS, and via our Bayesian inference technique we derive constraints on the neutrons stars' masses and radii. This method is applicable to other geodesic-frequency-based QPO models

Effects of Deoxycholylglycine, a Conjugated Secondary Bile Acid, on Myogenic Tone and Agonist-Induced Contraction in Rat Resistance Arteries

Bile acids (BAs) regulate cardiovascular function via diverse mechanisms. Although in both health and disease serum glycine-conjugated BAs are more abundant than taurine-conjugated BAs, their effects on myogenic tone (MT), a key determinant of systemic vascular resistance (SVR), have not been examined.Fourth-order mesenteric arteries (170-250 µm) isolated from Sprague-Dawley rats were pressurized at 70 mmHg and allowed to develop spontaneous constriction, i.e., MT. Deoxycholylglycine (DCG; 0.1-100 µM), a glycine-conjugated major secondary BA, induced reversible, concentration-dependent reduction of MT that was similar in endothelium-intact and -denuded arteries. DCG reduced the myogenic response to stepwise increase in pressure (20 to 100 mmHg). Neither atropine nor the combination of L-NAME (a NOS inhibitor) plus indomethacin altered DCG-mediated reduction of MT. K(+) channel blockade with glibenclamide (K(ATP)), 4-aminopyradine (K(V)), BaCl(2) (K(IR)) or tetraethylammonium (TEA, K(Ca)) were also ineffective. In Fluo-2-loaded arteries, DCG markedly reduced vascular smooth muscle cell (VSM) Ca(2+) fluorescence (∼50%). In arteries incubated with DCG, physiological salt solution (PSS) with high Ca(2+) (4 mM) restored myogenic response. DCG reduced vascular tone and VSM cytoplasmic Ca(2+) responses (∼50%) of phenylephrine (PE)- and Ang II-treated arteries, but did not affect KCl-induced vasoconstriction.In rat mesenteric resistance arteries DCG reduces pressure- and agonist-induced vasoconstriction and VSM cytoplasmic Ca(2+) responses, independent of muscarinic receptor, NO or K(+) channel activation. We conclude that BAs alter vasomotor responses, an effect favoring reduced SVR. These findings are likely pertinent to vascular dysfunction in cirrhosis and other conditions associated with elevated serum BAs

Black holes, gravitational waves and fundamental physics: a roadmap

The grand challenges of contemporary fundamental physics—dark matter, dark energy, vacuum energy, inflation and early universe cosmology, singularities and the hierarchy problem—all involve gravity as a key component. And of all gravitational phenomena, black holes stand out in their elegant simplicity, while harbouring some of the most remarkable predictions of General Relativity: event horizons, singularities and ergoregions. The hitherto invisible landscape of the gravitational Universe is being unveiled before our eyes: the historical direct detection of gravitational waves by the LIGO-Virgo collaboration marks the dawn of a new era of scientific exploration. Gravitational-wave astronomy will allow us to test models of black hole formation, growth and evolution, as well as models of gravitational-wave generation and propagation. It will provide evidence for event horizons and ergoregions, test the theory of General Relativity itself, and may reveal the existence of new fundamental fields. The synthesis of these results has the potential to radically reshape our understanding of the cosmos and of the laws of Nature. The purpose of this work is to present a concise, yet comprehensive overview of the state of the art in the relevant fields of research, summarize important open problems, and lay out a roadmap for future progress. This write-up is an initiative taken within the framework of the European Action on 'Black holes, Gravitational waves and Fundamental Physics'

Analysis of Factors that Regulate Asymmetric Cell Division in the alga Volvox carteri

Asymmetric cell divisions are critical for generating cellular diversity in multicellular organisms. Volvox carter

Analysis of Factors that Regulate Asymmetric Cell Division in the alga Volvox carteri

Asymmetric cell divisions are critical for generating cellular diversity in multicellular organisms. Volvox carter

Genome-wide Anaplasma phagocytophilum AnkA-DNA interactions are enriched in intergenic regions and gene promoters and correlate with infection-induced differential gene expression.

Anaplasma phagocytophilum, an obligate intracellular prokaryote, infects neutrophils and alters cardinal functions via reprogrammed transcription. Large contiguous regions of neutrophil chromosomes are differentially expressed during infection. Secreted A. phagocytophilum effector AnkA transits into the neutrophil or granulocyte nucleus to complex with DNA in heterochromatin across all chromosomes. AnkA binds to gene promoters to dampen cis-transcription and also has features of matrix attachment region (MAR)-binding proteins that regulate three-dimensional chromatin architecture and coordinate transcriptional programs encoded in topologically-associated chromatin domains. We hypothesize that identification of additional AnkA binding sites will better delineate how A. phagocytophilum infection results in reprogramming of the neutrophil genome. Using AnkA-binding ChIP-seq, we showed that AnkA binds broadly throughout all chromosomes in a reproducible pattern, especially at: i) intergenic regions predicted to be matrix attachment regions (MARs); ii) within predicted lamina-associated domains; and iii) at promoters ≤3,000 bp upstream of transcriptional start sites. These findings provide genome-wide support for AnkA as a regulator of cis-gene transcription. Moreover, the dominant mark of AnkA in distal intergenic regions known to be AT-enriched, coupled with frequent enrichment in the nuclear lamina, provides strong support for its role as a MAR-binding protein and genome re-organizer. AnkA must be considered a prime candidate to promote neutrophil reprogramming and subsequent functional changes that belie improved microbial fitness and pathogenicity

DCG reduces VSM Ca²⁺ in 4^th-order mesenteric arteries from rats with MT.

<p>Ca²⁺ fluorescence was measured in Fluo-2-loaded arteries before and after incubating with DCG 100 µM for 5 minutes. DCG reduced the arterial VSM Ca²⁺ fluorescence by ∼50%. (n = 3 arteries in each group).</p

Effect of DCG on agonist-induced vasoconstriction.

<p>(A) At 70 mmHg, the arteries developed spontaneous vasoconstriction that was further augmented by adding ANG II (0.1 µM). DCG attenuated ANG II-induced vasoconstriction. (n = 5 arteries). (B) An example tracing of the effect of DCG on ANG II-induced vasoconstriction. (C) At 70 mmHg, the arteries developed spontaneous vasoconstriction that was further augmented by adding PE (1 µM). DCG attenuated PE-induced vasoconstriction. (n = 12 arteries). (D) DCG (100 µM) did not alter 80 mM KCl-induced vasoconstriction. (n = 3 arteries). Asterisks indicate significant differences when compared to ANG II and PE alone.</p