HF-EPR, Raman, UV/VIS light spectroscopic, and DFT studies of the ribonucleotide reductase R2 tyrosyl radical from Epstein-Barr virus

Epstein-Barr virus (EBV) belongs to the gamma subfamily of herpes viruses, among the most common pathogenic viruses in humans worldwide. The viral ribonucleotide reductase small subunit (RNR R2) is involved in the biosynthesis of nucleotides, the DNA precursors necessary for viral replication, and is an important drug target for EBV. RNR R2 generates a stable tyrosyl radical required for enzymatic turnover. Here, the electronic and magnetic properties of the tyrosyl radical in EBV R2 have been determined by X-band and high-field/high-frequency electron paramagnetic resonance (EPR) spectroscopy recorded at cryogenic temperatures. The radical exhibits an unusually low g₁-tensor component at 2.0080, indicative of a positive charge in the vicinity of the radical. Consistent with these EPR results a relatively high C-O stretching frequency associated with the phenoxyl radical (at 1508 cm⁻¹) is observed with resonance Raman spectroscopy. In contrast to mouse R2, EBV R2 does not show a deuterium shift in the resonance Raman spectra. Thus, the presence of a water molecule as a hydrogen bond donor moiety could not be identified unequivocally. Theoretical simulations showed that a water molecule placed at a distance of 2.6 Å from the tyrosyl-oxygen does not result in a detectable deuterium shift in the calculated Raman spectra. UV/VIS light spectroscopic studies with metal chelators and tyrosyl radical scavengers are consistent with a more accessible dimetal binding/radical site and a lower affinity for Fe²⁺ in EBV R2 than in Escherichia coli R2. Comparison with previous studies of RNR R2s from mouse, bacteria, and herpes viruses, demonstrates that finely tuned electronic properties of the radical exist within the same RNR R2 Ia class.     

Shedding light on fish otolith biomineralization using a bioenergetic approach

Otoliths are biocalcified bodies connected to the sensory system in the inner ears of fish. Their layered, biorhythm-following formation provides individual records of the age, the individual history and the natural environment of extinct and living fish species. Such data are critical for ecosystem and fisheries monitoring. They however often lack validation and the poor understanding of biomineralization mechanisms has led to striking examples of misinterpretations and subsequent erroneous conclusions in fish ecology and fisheries management. Here we develop and validate a numerical model of otolith biomineralization. Based on a general bioenergetic theory, it disentangles the complex interplay between metabolic and temperature effects on biomineralization. This model resolves controversial issues and explains poorly understood observations of otolith formation. It represents a unique simulation tool to improve otolith interpretation and applications, and, beyond, to address the effects of both climate change and ocean acidification on other biomineralizing organisms such as corals and bivalves.     

Oral and fecal Campylobacter concisus strains perturb barrier function by apoptosis induction in HT-29/B6 intestinal epithelial cells

Campylobacter concisus infections of the gastrointestinal tract can be accompanied by diarrhea and inflammation, whereas colonization of the human oral cavity might have a commensal nature. We focus on the pathophysiology of C. concisus and the effects of different clinical oral and fecal C. concisus strains on human HT-29/B6 colon cells. Six oral and eight fecal strains of C. concisus were isolated. Mucus-producing HT-29/B6 epithelial monolayers were infected with the C. concisus strains. Transepithelial electrical resistance (R(t)) and tracer fluxes of different molecule size were measured in Ussing chambers. Tight junction (TJ) protein expression was determined by Western blotting, and subcellular TJ distribution was analyzed by confocal laser-scanning microscopy. Apoptosis induction was examined by TUNEL-staining and Western blot of caspase-3 activation. All strains invaded confluent HT-29/B6 cells and impaired epithelial barrier function, characterized by a time- and dose-dependent decrease in R(t) either after infection from the apical side but even more from the basolateral compartment. TJ protein expression changes were sparse, only in apoptotic areas of infected monolayers TJ proteins were redistributed. Solely the barrier-forming TJ protein claudin-5 showed a reduced expression level to 66±8% (P<0.05), by expression regulation from the gene. Concomitantly, Lactate dehydrogenase release was elevated to 3.1±0.3% versus 0.7±0.1% in control (P<0.001), suggesting cytotoxic effects. Furthermore, oral and fecal C. concisus strains elevated apoptotic events to 5-fold. C. concisus-infected monolayers revealed an increased permeability for 332 Da fluorescein (1.74±0.13 vs. 0.56±0.17 10(-6) cm/s in control, P<0.05) but showed no difference in permeability for 4 kDa FITC-dextran (FD-4). The same was true in camptothecin-exposed monolayers, where camptothecin was used for apoptosis induction.In conclusion, epithelial barrier dysfunction by oral and fecal C. concisus strains could mainly be assigned to apoptotic leaks together with moderate TJ changes, demonstrating a leak-flux mechanism that parallels the clinical manifestation of diarrhea.     

Testing the water-energy theory on American palms (Arecaceae) using geographically weighted regression

Water and energy have emerged as the best contemporary environmental correlates of broad-scale species richness patterns. A corollary hypothesis of water-energy dynamics theory is that the influence of water decreases and the influence of energy increases with absolute latitude. We report the first use of geographically weighted regression for testing this hypothesis on a continuous species richness gradient that is entirely located within the tropics and subtropics. The dataset was divided into northern and southern hemispheric portions to test whether predictor shifts are more pronounced in the less oceanic northern hemisphere. American palms (Arecaceae, n = 547 spp.), whose species richness and distributions are known to respond strongly to water and energy, were used as a model group. The ability of water and energy to explain palm species richness was quantified locally at different spatial scales and regressed on latitude. Clear latitudinal trends in agreement with water-energy dynamics theory were found, but the results did not differ qualitatively between hemispheres. Strong inherent spatial autocorrelation in local modeling results and collinearity of water and energy variables were identified as important methodological challenges. We overcame these problems by using simultaneous autoregressive models and variation partitioning. Our results show that the ability of water and energy to explain species richness changes not only across large climatic gradients spanning tropical to temperate or arctic zones but also within megathermal climates, at least for strictly tropical taxa such as palms. This finding suggests that the predictor shifts are related to gradual latitudinal changes in ambient energy (related to solar flux input) rather than to abrupt transitions at specific latitudes, such as the occurrence of frost.     

Sensory coding by cerebellar mossy fibres through inhibition-driven phase resetting and synchronisation

Temporal coding of spike-times using oscillatory mechanisms allied to spike-time dependent plasticity could represent a powerful mechanism for neuronal communication. However, it is unclear how temporal coding is constructed at the single neuronal level. Here we investigate a novel class of highly regular, metronome-like neurones in the rat brainstem which form a major source of cerebellar afferents. Stimulation of sensory inputs evoked brief periods of inhibition that interrupted the regular firing of these cells leading to phase-shifted spike-time advancements and delays. Alongside phase-shifting, metronome cells also behaved as band-pass filters during rhythmic sensory stimulation, with maximal spike-stimulus synchronisation at frequencies close to the idiosyncratic firing frequency of each neurone. Phase-shifting and band-pass filtering serve to temporally align ensembles of metronome cells, leading to sustained volleys of near-coincident spike-times, thereby transmitting synchronised sensory information to downstream targets in the cerebellar cortex.     

Impact of non-HIV and HIV risk factors on survival in HIV-infected patients on HAART: a population-based nationwide cohort study

Background

We determined the impact of three factors on mortality in HIV-infected patients who had been on highly active antiretroviral therapy (HAART) for at least one year: (1) insufficient response to (HAART) and presence of AIDS-defining diseases, (2) comorbidity, and (3) drug and alcohol abuse and compared the mortality to that of the general population.

Methodology/Principal Findings

In a Danish nationwide, population-based cohort study, we used population based registries to identify (1) all Danish HIV-infected patients who started HAART in the period 1 January 1998–1 July 2009, and (2) a comparison cohort of individuals matched on date of birth and gender (N = 2,267 and 9,068, respectively). Study inclusion began 1 year after start of HAART. Patients were categorised hierarchically in four groups according to the three risk factors, which were identified before study inclusion. The main outcome measure was probability of survival from age 25 to 65 years. The probability of survival from age 25 to age 65 was substantially lower in HIV patients [0.48 (95% confidence interval (CI) 0.42–0.55)] compared to the comparison cohort [0.88 (0.86 to 0.90)]. However, in HIV patients with no risk factors (N = 871) the probability of survival was equivalent to that of the general population [0.86 (95% CI 0.77–0.92)]. In contrast, the probability of survival was 0.58 in patients with HIV risk factors (N = 704), 0.30 in patients with comorbidities (N = 479), and 0.03 in patients with drug or alcohol abuse (N = 313).

Conclusions

The increased risk of death in HIV-infected individuals is mainly attributable to risk factors that can be identified prior to or in the initial period of antiretroviral treatment. Mortality in patients without risk factors on a successful HAART is almost identical to that of the non–HIV-infected population.     

Circadian clocks in mouse and human CD4+ T cells

Though it has been shown that immunological functions of CD4+ T cells are time of day-dependent, the underlying molecular mechanisms remain largely obscure. To address the question whether T cells themselves harbor a functional clock driving circadian rhythms of immune function, we analyzed clock gene expression by qPCR in unstimulated CD4+ T cells and immune responses of PMA/ionomycin stimulated CD4+ T cells by FACS analysis purified from blood of healthy subjects at different time points throughout the day. Molecular clock as well as immune function was further analyzed in unstimulated T cells which were cultured in serum-free medium with circadian clock reporter systems. We found robust rhythms of clock gene expression as well as, after stimulation, IL-2, IL-4, IFN-γ production and CD40L expression in freshly isolated CD4+ T cells. Further analysis of IFN-γ and CD40L in cultivated T cells revealed that these parameters remain rhythmic in vitro. Moreover, circadian luciferase reporter activity in CD4+ T cells and in thymic sections from PER2::LUCIFERASE reporter mice suggest that endogenous T cell clock rhythms are self-sustained under constant culture conditions. Microarray analysis of stimulated CD4+ T cell cultures revealed regulation of the NF-κB pathway as a candidate mechanism mediating circadian immune responses. Collectively, these data demonstrate for the first time that CD4+ T cell responses are regulated by an intrinsic cellular circadian oscillator capable of driving rhythmic CD4+ T cell immune responses.     

Copy number variation in patients with disorders of sex development due to 46,XY gonadal dysgenesis

Disorders of sex development (DSD), ranging in severity from mild genital abnormalities to complete sex reversal, represent a major concern for patients and their families. DSD are often due to disruption of the genetic programs that regulate gonad development. Although some genes have been identified in these developmental pathways, the causative mutations have not been identified in more than 50% 46,XY DSD cases. We used the Affymetrix Genome-Wide Human SNP Array 6.0 to analyse copy number variation in 23 individuals with unexplained 46,XY DSD due to gonadal dysgenesis (GD). Here we describe three discrete changes in copy number that are the likely cause of the GD. Firstly, we identified a large duplication on the X chromosome that included DAX1 (NR0B1). Secondly, we identified a rearrangement that appears to affect a novel gonad-specific regulatory region in a known testis gene, SOX9. Surprisingly this patient lacked any signs of campomelic dysplasia, suggesting that the deletion affected expression of SOX9 only in the gonad. Functional analysis of potential SRY binding sites within this deleted region identified five putative enhancers, suggesting that sequences additional to the known SRY-binding TES enhancer influence human testis-specific SOX9 expression. Thirdly, we identified a small deletion immediately downstream of GATA4, supporting a role for GATA4 in gonad development in humans. These CNV analyses give new insights into the pathways involved in human gonad development and dysfunction, and suggest that rearrangements of non-coding sequences disturbing gene regulation may account for significant proportion of DSD cases.     

Tasting soil fungal diversity with earth tongues: phylogenetic test of SATé alignments for environmental ITS data

An abundance of novel fungal lineages have been indicated by DNA sequencing of the nuclear ribosomal ITS region from environmental samples such as soil and wood. Although phylogenetic analysis of these novel lineages is a key component of unveiling the structure and diversity of complex communities, such analyses are rare for environmental ITS data due to the difficulties of aligning this locus across significantly divergent taxa. One potential approach to this issue is simultaneous alignment and tree estimation. We targeted divergent ITS sequences of the earth tongue fungi (Geoglossomycetes), a basal class in the Ascomycota, to assess the performance of SATé, recent software that combines progressive alignment and tree building. We found that SATé performed well in generating high-quality alignments and in accurately estimating the phylogeny of earth tongue fungi. Drawing from a data set of 300 sequences of earth tongues and progressively more distant fungal lineages, 30 insufficiently identified ITS sequences from the public sequence databases were assigned to the Geoglossomycetes. The association between earth tongues and plants has been hypothesized for a long time, but hard evidence is yet to be collected. The ITS phylogeny showed that four ectomycorrhizal isolates shared a clade with Geoglossum but not with Trichoglossum earth tongues, pointing to the significant potential inherent to ecological data mining of environmental samples. Environmental sampling holds the key to many focal questions in mycology, and simultaneous alignment and tree estimation, as performed by SATé, can be a highly efficient companion in that pursuit.