Codon usage patterns in Nematoda: analysis based on over 25 million codons in thirty-two species

Background  

Codon usage has direct utility in molecular characterization of species and is also a marker for molecular evolution. To understand codon usage within the diverse phylum Nematoda, we analyzed a total of 265,494 expressed sequence tags (ESTs) from 30 nematode species. The full genomes of Caenorhabditis elegans and C. briggsae were also examined. A total of 25,871,325 codons were analyzed and a comprehensive codon usage table for all species was generated. This is the first codon usage table available for 24 of these organisms.     

Characteristics of nest soil, but not geographic origin, influence cold hardiness of hatchling painted turtles

We investigated environmental factors influencing cold hardiness in hatchling painted turtles (Chrysemys picta) indigenous to northeastern Indiana and the Sandhills of west-central Nebraska. In both locations, hatchlings overwinter in their natal nests. Survival of hatchlings chilled to minimum temperatures between -2.5 and -6.0 degrees C inside explanted natal nests ranged from 30 to 100%. Mortality likely was caused by freezing of the turtles that was induced by contact with ice nuclei in the surrounding soil. Susceptibility to inoculative freezing was strongly influenced by moisture content (7.5-25%, w/w) of the frozen soil in which hatchlings were cooled. When chilled in soil containing 15% moisture, turtles from Indiana resisted inoculative freezing better than hatchlings from Nebraska, but this variation was due to physical characteristics of the soils indigenous to each locale rather than genetic differences between populations. Soil in which the Indiana turtles nested contained relatively higher amounts of clay and organic matter, and bound more moisture, than the loamy sand at the Nebraska site. Soil collected from both locales contained potent ice nuclei that may constrain supercooling of the hatchlings, even in the absence of soil moisture. In addition to temperature and precipitation, local and regional variation in soils is an important determinant of overwintering survival of hatchling C. picta.     

Silicon modifies root anatomy, and uptake and subcellular distribution of cadmium in young maize plants

Background and Aims

Silicon (Si) has been shown to ameliorate the negative influence of cadmium (Cd) on plant growth and development. However, the mechanism of this phenomenon is not fully understood. Here we describe the effect of Si on growth, and uptake and subcellular distribution of Cd in maize plants in relation to the development of root tissues.

Methods

Young maize plants (Zea mays) were cultivated for 10 d hydroponically with 5 or 50 µm Cd and/or 5 mm Si. Growth parameters and the concentrations of Cd and Si were determined in root and shoot by atomic absorption spectrometry or inductively coupled plasma mass spectroscopy. The development of apoplasmic barriers (Casparian bands and suberin lamellae) and vascular tissues in roots were analysed, and the influence of Si on apoplasmic and symplasmic distribution of ¹⁰⁹Cd applied at 34 nm was investigated between root and shoot.

Key Results

Si stimulated the growth of young maize plants exposed to Cd and influenced the development of Casparian bands and suberin lamellae as well as vascular tissues in root. Si did not affect the distribution of apoplasmic and symplasmic Cd in maize roots, but considerably decreased symplasmic and increased apoplasmic concentration of Cd in maize shoots.

Conclusions

Differences in Cd uptake of roots and shoots are probably related to the development of apoplasmic barriers and maturation of vascular tissues in roots. Alleviation of Cd toxicity by Si might be attributed to enhanced binding of Cd to the apoplasmic fraction in maize shoots.     

The effects of angiotensin II on the coupled microstructural and biomechanical response of C57BL/6 mouse aorta

RationaleAbdominal aortic aneurysm (AAA) is a complex disease that leads to a localized dilation of the infrarenal aorta, the rupture of which is associated with significant morbidity and mortality. Animal models of AAA can be used to study how changes in the microstructural and biomechanical behavior of aortic tissues develop as disease progresses in these animals. We chose here to investigate the effect of angiotensin II (AngII) in C57BL/6 mice as a first step towards understanding how such changes occur in the established ApoE^?/? AngII infused mouse model of AAA.ObjectiveThe objective of this study was to utilize a recently developed device in our laboratory to determine how the microstructural and biomechanical properties of AngII-infused C57BL/6 wildtype mouse aorta change following 14 days of AngII infusion.MethodsC57BL/6 wildtype mice were infused with either saline or AngII for 14 day. Aortas were excised and tested using a device capable of simultaneously characterizing the biaxial mechanical response and load-dependent (unfixed, unfrozen) extracellular matrix organization of mouse aorta (using multiphoton microscopy). Peak strains and stiffness values were compared across experimental groups, and both datasets were fit to a Fung-type constitutive model. The mean mode and full width at half maximum (FWHM) of fiber histograms from two photon microscopy were quantified in order to assess the preferred fiber distribution and degree of fiber splay, respectively.ResultsThe axial stiffness of all mouse aorta was found to be an order of magnitude larger than the circumferential stiffness. The aortic diameter was found to be significantly increased for the AngII infused mice as compared to saline infused control (p=0.026). Aneurysm, defined as a percent increase in maximum diameter of 30% (defined with respect to saline control), was found in 3 of the 6 AngII infused mice. These three mice displayed adventitial collagen that lacked characteristic fiber crimp. The biomechanical response in the AngII infused mice showed significantly reduced circumferential compliance. We also noticed that the ability of the adventitial collagen fibers in AngII infused mice to disperse in reaction to circumferential loading was suppressed.ConclusionsCollagen remodeling is present following 14 days of AngII infusion in C57BL/6 mice. Aneurysmal development occurred in 50% of our AngII infused mice, and these dilatations were accompanied with adventitial collagen remodeling and decreased circumferential compliance.     

Molecular pathogenesis of a new glycogenosis caused by a glycogenin-1 mutation

Glycogenin-1 initiates the glycogen synthesis in skeletal muscle by the autocatalytic formation of a short oligosaccharide at tyrosine 195. Glycogenin-1 catalyzes both the glucose-O-tyrosine linkage and the α1,4 glucosidic bonds linking the glucose molecules in the oligosaccharide. We recently described a patient with glycogen depletion in skeletal muscle as a result of a non-functional glycogenin-1. The patient carried a Thr83Met substitution in glycogenin-1. In this study we have investigated the importance of threonine 83 for the catalytic activity of glycogenin-1. Non-glucosylated glycogenin-1 constructs, with various amino acid substitutions in position 83 and 195, were expressed in a cell-free expression system and autoglucosylated in vitro. The autoglucosylation was analyzed by gel-shift on western blot, incorporation of radiolabeled UDP-(14)C-glucose and nano-liquid chromatography with tandem mass spectrometry (LC/MS/MS). We demonstrate that glycogenin-1 with the Thr83Met substitution is unable to form the glucose-O-tyrosine linkage at tyrosine 195 unless co-expressed with the catalytically active Tyr195Phe glycogenin-1. Our results explain the glycogen depletion in the patient expressing only Thr83Met glycogenin-1 and why heterozygous carriers without clinical symptoms show a small proportion of unglucosylated glycogenin-1.     

What can phylogeographic breaks in the livebearer Poeciliopsis turrubarensis tell about fish community boundaries in western Costa Rica?

This study asks if the geographic boundary delineating two fish communities in western Costa Rica is congruent with a phylogeographic break in a single widespread fish species Poeciliopsis turrubarensis (Poeciliidae) that spans this area. Such congruence would suggest that a common historical event (e.g. geological or climatic) could be responsible for both patterns. It was found that there was a shared break across a region in central Costa Rica suggesting a common cause may be responsible for both the abrupt shift in fish community composition and the genetic break in P. turrubarensis.     

Cellular context and multiple channel domains determine cAMP sensitivity of HCN4 channels: Ligand-independent relief of autoinhibition in HCN4

Hyperpolarization-activated, cyclic nucleotide–sensitive (HCN) channels produce the I_f and I_h currents, which are critical for cardiac pacemaking and neuronal excitability, respectively. HCN channels are modulated by cyclic AMP (cAMP), which binds to a conserved cyclic nucleotide–binding domain (CNBD) in the C terminus. The unliganded CNBD has been shown to inhibit voltage-dependent gating of HCNs, and cAMP binding relieves this “autoinhibition,” causing a depolarizing shift in the voltage dependence of activation. Here we report that relief of autoinhibition can occur in the absence of cAMP in a cellular context- and isoform-dependent manner: when the HCN4 isoform was expressed in Chinese hamster ovary (CHO) cells, the basal voltage dependence was already shifted to more depolarized potentials and cAMP had no further effect on channel activation. This “pre-relief” of autoinhibition was specific both to HCN4 and to CHO cells; cAMP shifted the voltage dependence of HCN2 in CHO cells and of HCN4 in human embryonic kidney (HEK) cells. The pre-relief phenotype did not result from different concentrations of soluble intracellular factors in CHO and HEK cells, as it persisted in excised cell-free patches. Likewise, it did not arise from a failure of cAMP to bind to the CNBD of HCN4 in CHOs, as indicated by cAMP-dependent slowing of deactivation. Instead, a unique ∼300–amino acid region of the distal C terminus of HCN4 (residues 719–1012, downstream of the CNBD) was found to be necessary, but not sufficient, for the depolarized basal voltage dependence and cAMP insensitivity of HCN4 in CHO cells. Collectively, these data suggest a model in which multiple HCN4 channel domains conspire with membrane-associated intracellular factors in CHO cells to relieve autoinhibition in HCN4 channels in the absence of cAMP. These findings raise the possibility that such ligand-independent regulation could tune the activity of HCN channels and other CNBD-containing proteins in many physiological systems.     

Total sleep deprivation alters cardiovascular reactivity to acute stressors in humans

Exaggerated cardiovascular reactivity to mental stress (MS) and cold pressor test (CPT) has been linked to increased risk of cardiovascular disease. Recent epidemiological studies identify sleep deprivation as an important risk factor for hypertension, yet the relations between sleep deprivation and cardiovascular reactivity remain equivocal. We hypothesized that 24-h total sleep deprivation (TSD) would augment cardiovascular reactivity to MS and CPT and blunt the MS-induced forearm vasodilation. Because the associations between TSD and hypertension appear to be stronger in women, a secondary aim was to probe for sex differences. Mean arterial pressure (MAP), heart rate (HR), and muscle sympathetic nerve activity (MSNA) were recorded during MS and CPT in 28 young, healthy subjects (14 men and 14 women) after normal sleep (NS) and 24-h TSD (randomized, crossover design). Forearm vascular conductance (FVC) was recorded during MS. MAP, FVC, and MSNA (n = 10) responses to MS were not different between NS and TSD (condition × time, P > 0.05). Likewise, MAP and MSNA (n = 6) responses to CPT were not different between NS and TSD (condition × time, P > 0.05). In contrast, increases in HR during both MS and CPT were augmented after TSD (condition × time, P ≤ 0.05), and these augmented HR responses persisted during both recoveries. When analyzed for sex differences, cardiovascular reactivity to MS and CPT was not different between sexes (condition × time × sex, P > 0.05). We conclude that TSD does not significantly alter MAP, MSNA, or forearm vascular responses to MS and CPT. The augmented tachycardia responses during and after both acute stressors provide new insight regarding the emerging links among sleep deprivation, stress, and cardiovascular risk.     

Two systems for targeted gene deletion in Coxiella burnetii

Coxiella burnetii is a ubiquitous zoonotic bacterial pathogen and the cause of human acute Q fever, a disabling influenza-like illness. C. burnetii's former obligate intracellular nature significantly impeded the genetic characterization of putative virulence factors. However, recent host cell-free (axenic) growth of the organism has enabled development of shuttle vector, transposon, and inducible gene expression technologies, with targeted gene inactivation remaining an important challenge. In the present study, we describe two methods for generating targeted gene deletions in C. burnetii that exploit pUC/ColE1 ori-based suicide plasmids encoding sacB for positive selection of mutants. As proof of concept, C. burnetii dotA and dotB, encoding structural components of the type IVB secretion system (T4BSS), were selected for deletion. The first method exploited Cre-lox-mediated recombination. Two suicide plasmids carrying different antibiotic resistance markers and a loxP site were integrated into 5' and 3' flanking regions of dotA. Transformation of this strain with a third suicide plasmid encoding Cre recombinase resulted in the deletion of dotA under sucrose counterselection. The second method utilized a loop-in/loop-out strategy to delete dotA and dotB. A single suicide plasmid was first integrated into 5' or 3' target gene flanking regions. Resolution of the plasmid cointegrant by a second crossover event under sucrose counterselection resulted in gene deletion that was confirmed by PCR and Southern blot. ΔdotA and ΔdotB mutants failed to secrete T4BSS substrates and to productively infect host cells. The repertoire of C. burnetii genetic tools now allows ready fulfillment of molecular Koch's postulates for suspected virulence genes.