ATP and its N<Superscript>6</Superscript>-substituted analogues: parameterization,molecular dynamics simulation and conformational analysis

In this work we used a combination of classical molecular dynamics and simulated annealing techniques to shed more light on the conformational flexibility of 12 adenosine triphosphate (ATP) analogues in a water environment. We present simulations in AMBER force field for ATP and 12 published analogues [Shah et al. (1997) Proc Natl Acad Sci USA 94: 3565–3570]. The calculations were carried out using the generalized Born (GB) solvation model in the presence of the cation Mg²⁺. The ion was placed at a close distance (2 Å) from the charged oxygen atoms of the beta and gamma phosphate groups of the ?3 negatively charged ATP analogue molecules. Analysis of the results revealed the distribution of inter-proton distances H8–H1′ and H8–H2′ versus the torsion angle ψ (C4–N9-C1′–O4′) for all conformations of ATP analogues. There are two gaps in the distribution of torsion angle ψ values: the first is between ?30 and 30 degrees and is described by cis-conformation; and the second is between 90 and 175 degrees, which mostly covers a region of anti conformation. Our results compare favorably with results obtained in experimental assays [Jiang and Mao (2002) Polyhedron 21:435–438].

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Figure Dihedral O4′–C1′–N9–C4 angle dependence on inter-proton distances H8–H1′ (crosses) and H8–H2′ (dots) measured for ATP

     

Therapeutic siRNA silencing in inflammatory monocytes in mice

Excessive and prolonged activity of inflammatory monocytes is a hallmark of many diseases with an inflammatory component. In such conditions, precise targeting of these cells could be therapeutically beneficial while sparing many essential functions of the innate immune system, thus limiting unwanted effects. Inflammatory monocytes-but not the noninflammatory subset-depend on the chemokine receptor CCR2 for localization to injured tissue. Here we present an optimized lipid nanoparticle and a CCR2-silencing short interfering RNA that, when administered systemically in mice, show rapid blood clearance, accumulate in spleen and bone marrow, and localize to monocytes. Efficient degradation of CCR2 mRNA in monocytes prevents their accumulation in sites of inflammation. Specifically, the treatment attenuates their number in atherosclerotic plaques, reduces infarct size after coronary artery occlusion, prolongs normoglycemia in diabetic mice after pancreatic islet transplantation, and results in reduced tumor volumes and lower numbers of tumor-associated macrophages.     

Trans fat diet causes decreased brood size and shortened lifespan in Caenorhabditis elegans delta-6-desaturase mutant fat-3

Trans-fatty acids (TFAs) enter the diet through industrial processes and can cause adverse human health effects. The present study was aimed to examine the effects of dietary cis- and trans-fatty acids on the model organism Caenorhabditis elegans. Cis- or trans-18:1n9 triglycerides (25 μM) caused no apparent changes in the numbers of viable progeny of wild-type N2 animals. However, in fat-3 mutants lacking delta-6-desaturase, the trans-isomer caused modest decreases in lifespan and progeny after three generations. Long-chain polyunsaturated fatty acids (PUFA) profiles were significantly altered in fat-3 mutants compared to wild type but were not altered after exposure to dietary cis- or trans-18:1n9. Genome-wide expression analysis of fat-3 mutants revealed hundreds of changes. Several genes involved in fat metabolism (acs-2, fat-7, mdt-15) were significantly increased by cis- or trans-18:1n9 without discrimination between isomers. These results provide support for the hypothesis that dietary trans fats are detrimental to development and aging.     

Microbial community analysis and identification of alternative host-specific fecal indicators in fecal and river water samples using pyrosequencing

It is important to know the comprehensive microbial communities of fecal pollution sources and receiving water bodies for microbial source tracking. Pyrosequencing targeting the V1-V3 hypervariable regions of the 16S rRNA gene was used to investigate the characteristics of bacterial and Bacteroidales communities in major fecal sources and river waters. Diversity analysis indicated that cow feces had the highest diversities in the bacterial and Bacteroidales group followed by the pig sample, with human feces having the lowest value. The Bacteroidales, one of the potential fecal indicators, totally dominated in the fecal samples accounting for 31%-52% of bacterial sequences, but much less (0.6%) in the river water. Clustering and Venn diagram analyses showed that the human sample had a greater similarity to the pig sample in the bacterial and Bacteroidales communities than to samples from other hosts. Traditional fecal indicators, i.e., Escherichia coli, were detected in the human and river water samples at very low rates and Clostridium perfringens and enterococci were not detected in any samples. Besides the Bacteroidales group, some microorganisms detected in the specific hosts, i.e., Parasutterella excrementihominis, Veillonella sp., Dialister invisus, Megamonas funiformis, and Ruminococcus lactaris for the human and Lactobacillus amylovorus and Atopostipes sp. for the pig, could be used as potential host-specific fecal indicators. These microorganisms could be used as multiple fecal indicators that are not dependent on the absence or presence of a single indicator. Monitoring for multiple indicators that are highly abundant and host-specific would greatly enhance the effectiveness of fecal pollution source tracking.     

Minimal effect of acute caffeine ingestion on intense resistance training performance

The primary aim of the study was to determine the efficacy of acute caffeine intake to enhance intense resistance training performance. Fourteen resistance-trained men (age and body mass = 23.1 ± 1.1 years and 83.4 ± 13.2 kg, respectively) who regularly consumed caffeine ingested caffeine (6 mg · kg(-1)) or placebo 1 hour before completion of 4 sets of barbell bench press, leg press, bilateral row, and barbell shoulder press to fatigue at 70-80% 1-repetition maximum. Two minutes of rest was allotted between sets. Saliva samples were obtained to assess caffeine concentration. The number of repetitions completed per set and total weight lifted were recorded as indices of performance. Two-way analysis of variance with repeated measures was used to examine differences in performance across treatment and sets. Compared to placebo, there was a small but significant effect (p < 0.05) of acute caffeine intake on repetitions completed for the leg press but not for upper-body exercise (p > 0.05). Total weight lifted across sets was similar (p > 0.05) with caffeine (22,409.5 ± 3,773.2 kg) vs. placebo (21,185.7 ± 4,655.4 kg), yet there were 9 'responders' to caffeine, represented by a meaningful increase in total weight lifted with caffeine vs. placebo. Any ergogenic effect of caffeine on performance of fatiguing, total-body resistance training appears to be of limited practical significance. Additional research is merited to elucidate interindividual differences in caffeine-mediated improvements in performance.     

Hypoxic regulation of mesenchymal stem cell migration: the role of RhoA and HIF-1α

A variety of pathologies such as skeletal fracture, neoplasia and inflammation compromise tissue perfusion and thereby decrease tissue oxygen tension. We and others have demonstrated that hypoxia is a potent stimulant for MSC (mesenchymal stem cell) recruitment and differentiation, yet to date little research has focused on the effects of oxygen tension on MSC migration. In the present study, we examined the effects of hypoxia and the potential role of the GTPase RhoA and HIF-1α (hypoxia-inducible factor 1α) on MSC migration. Our results demonstrate that hypoxia decreases MSC migration through an HIF-1α and RhoA-mediated pathway. The active GTP-bound form of RhoA was reduced in 1% oxygen, whereas activation of RhoA under hypoxic conditions rescued migration. Furthermore, stabilization of HIF-1α under normoxic conditions attenuated cell migration similar to that of hypoxia. These results suggest that hypoxia negatively affects MSC migration by regulating activation of GTPases. These results highlight the importance of oxygen in regulating the recruitment of progenitor cells to areas of ischaemic tissue damage.     

A nonerythropoietic peptide that mimics the 3D structure of erythropoietin reduces organ injury/dysfunction and inflammation in experimental hemorrhagic shock

Recent studies have shown that erythropoietin, critical for the differentiation and survival of erythrocytes, has cytoprotective effects in a wide variety of tissues, including the kidney and lung. However, erythropoietin has been shown to have a serious side effect-an increase in thrombovascular effects. We investigated whether pyroglutamate helix B-surface peptide (pHBSP), a nonerythropoietic tissue-protective peptide mimicking the 3D structure of erythropoietin, protects against the organ injury/ dysfunction and inflammation in rats subjected to severe hemorrhagic shock (HS). Mean arterial blood pressure was reduced to 35 ± 5 mmHg for 90 min followed by resuscitation with 20 mL/kg Ringer Lactate for 10 min and 50% of the shed blood for 50 min. Rats were euthanized 4 h after the onset of resuscitation. pHBSP was administered 30 min or 60 min into resuscitation. HS resulted in significant organ injury/dysfunction (renal, hepatic, pancreas, neuromuscular, lung) and inflammation (lung). In rats subjected to HS, pHBSP significantly attenuated (i) organ injury/dysfunction (renal, hepatic, pancreas, neuromuscular, lung) and inflammation (lung), (ii) increased the phosphorylation of Akt, glycogen synthase kinase-3β and endothelial nitric oxide synthase, (iii) attenuated the activation of nuclear factor (NF)-κB and (iv) attenuated the increase in p38 and extracellular signal-regulated kinase (ERK)1/2 phosphorylation. pHBSP protects against multiple organ injury/dysfunction and inflammation caused by severe hemorrhagic shock by a mechanism that may involve activation of Akt and endothelial nitric oxide synthase, and inhibition of glycogen synthase kinase-3β and NF-κB.     

Subunit organization in the Dam1 kinetochore complex and its ring around microtubules

All eukaryotic cells must segregate their chromosomes equally between two daughter cells at each division. This process needs to be robust, as errors in the form of loss or gain of genetic material have catastrophic effects on viability. Chromosomes are captured, aligned, and segregated to daughter cells via interaction with spindle microtubules mediated by the kinetochore. In Saccharomyces cerevisiae one microtubule attaches to each kinetochore, requiring extreme processivity from this single connection. The yeast Dam1 complex, an essential component of the outer kinetochore, forms rings around microtubules and in vitro recapitulates much of the functionality of a kinetochore-microtubule attachment. To understand the mechanism of the Dam1 complex at the kinetochore, we must know how it binds to microtubules, how it assembles into rings, and how assembly is regulated. We used electron microscopy to map several subunits within the structure of the Dam1 complex and identify the organization of Dam1 complexes within the ring. Of importance, new data strongly support a more passive role for the microtubule in Dam1 ring formation. Integrating this information with previously published data, we generated a structural model for the Dam1 complex assembly that advances our understanding of its function and will direct future experiments.