Novel positive-sense, single-stranded RNA (+ssRNA) virus with di-cistronic genome from intestinal content of freshwater carp (Cyprinus carpio)

A novel positive-sense, single-stranded RNA (+ssRNA) virus (Halastavi árva RNA virus, HalV; {"type":"entrez-nucleotide","attrs":{"text":"JN000306","term_id":"347602590","term_text":"JN000306"}}JN000306) with di-cistronic genome organization was serendipitously identified in intestinal contents of freshwater carps (Cyprinus carpio) fished by line-fishing from fishpond “Lőrinte halastó” located in Veszprém County, Hungary. The complete nucleotide (nt) sequence of the genomic RNA is 9565 nt in length and contains two long - non-in-frame - open reading frames (ORFs), which are separated by an intergenic region. The ORF1 (replicase) is preceded by an untranslated sequence of 827 nt, while an untranslated region of 139 nt follows the ORF2 (capsid proteins). The deduced amino acid (aa) sequences of the ORFs showed only low (less than 32%) and partial similarity to the non-structural (2C-like helicase, 3C-like cystein protease and 3D-like RNA dependent RNA polymerase) and structural proteins (VP2/VP4/VP3) of virus families in Picornavirales especially to members of the viruses with dicistronic genome. Halastavi árva RNA virus is present in intestinal contents of omnivorous freshwater carps but the origin and the host species of this virus remains unknown. The unique viral sequence and the actual position indicate that Halastavi árva RNA virus seems to be the first member of a new di-cistronic ssRNA virus. Further studies are required to investigate the specific host species (and spectrum), ecology and role of Halastavi árva RNA virus in the nature.     

LC8 dynein light chain (DYNLL1) binds to the C-terminal domain of ATM-interacting protein (ATMIN/ASCIZ) and regulates its subcellular localization

LC8 dynein light chain (now termed DYNLL1 and DYNLL2 in mammals), a dimeric 89 amino acid protein, is a component of the dynein multi-protein complex. However a substantial amount of DYNLL1 is not associated to microtubules and it can thus interact with dozens of cellular and viral proteins that display well-defined, short linear motifs. Using DYNLL1 as bait in a yeast two-hybrid screen of a human heart library we identified ATMIN, an ATM kinase-interacting protein, as a DYNLL1-binding partner. Interestingly, ATMIN displays at least 18 SQ/TQ motifs in its sequence and DYNLL1 is known to bind to proteins with KXTQT motifs. Using pepscan and yeast two-hybrid techniques we show that DYNLL1 binds to multiple SQ/TQ motifs present in the carboxy-terminal domain of ATMIN. Recombinant expression and purification of the DYNLL1-binding region of ATMIN allowed us to obtain a polypeptide with an apparent molecular mass in gel filtration close to 400 kDa that could bind to DYNLL1 in vitro. The NMR data-driven modelled complexes of DYNLL1 with two selected ATMIN peptides revealed a similar mode of binding to that observed between DYNLL1 and other peptide targets. Remarkably, co-expression of mCherry-DYNLL1 and GFP-ATMIN mutually affected intracellular protein localization. In GFP-ATMIN expressing-cells DNA damage induced efficiently nuclear foci formation, which was partly impeded by the presence of mCherry-DYNLL1. Thus, our results imply a potential cellular interference between DYNLL1 and ATMIN functions.     

Apical localization of PMCA2w/b is enhanced in terminally polarized MDCK cells

The “w” splice forms of PMCA2 localize to distinct membrane compartments such as the apical membrane of the lactating mammary epithelium, the stereocilia of inner ear hair cells or the post-synaptic density of hippocampal neurons. Previous studies indicated that PMCA2w/b was not fully targeted to the apical domain of MDCK cells but distributed more evenly to the lateral and apical membrane compartments. Overexpression of the apical scaffold protein NHERF2, however, greatly increased the amount of the pump in the apical membrane of these epithelial cells. We generated a stable MDCK cell line expressing non-tagged, full-length PMCA2w/b to further study the localization and function of this protein. Here we demonstrate that PMCA2w/b is highly active and shows enhanced apical localization in terminally polarized MDCK cells grown on semi-permeable filters. Reversible surface biotinylation combined with confocal microscopy of fully polarized cells show that the pump is stabilized in the apical membrane via the apical membrane cytoskeleton with the help of endogenous NHERF2 and ezrin. Disruption of the actin cytoskeleton removed the pump from the apical actin patches without provoking its internalization. Our data suggest that full polarization is a prerequisite for proper positioning of the PMCA2w variants in the apical membrane domain of polarized cells.     

Conformational preferences in diglycosyl disulfides: NMR and molecular modeling studies

The conformations of several β1→β1′ diglycosyl disulfides were investigated by NMR and computational methods. Experimental data, such as NOEs, proton–proton and proton–carbon-13 coupling constants, measured for solutions in DMSO, are in good agreement with values obtained by MD simulations in explicit DMSO. The disulfide torsion angles (C1–S–S–C1′) preferentially sample values close to either +90° or −90° (+g or −g) and appear as the main metric that determines the conformational behavior of these glycomimetics. There is more conformational freedom around the C1–S and C1′–S′ bonds (Φ and Ω torsions, respectively) and population cluster analysis allowed to identify up to four allowed conformational regions for each of the +g or −g forms. Population analysis of the hydroxylic group rotamers, based on proton–proton and proton–carbon-13 couplings as well as on calculated hydrogen bonding statistics, did not reveal any significant intramolecular hydrogen bonds in DMSO solution.     

Temperature,but Not Available Energy,Affects the Expression of a Sexually Selected Ultraviolet (UV) Colour Trait in Male European Green Lizards

Background

Colour signals are widely used in intraspecific communication and often linked to individual fitness. The development of some pigment-based (e.g. carotenoids) colours is often environment-dependent and costly for the signaller, however, for structural colours (e.g. ultraviolet [UV]) this topic is poorly understood, especially in terrestrial ectothermic vertebrates.

Methodology/Principal Findings

In a factorial experiment, we studied how available energy and time at elevated body temperature affects the annual expression of the nuptial throat colour patch in male European green lizards (Lacerta viridis) after hibernation and before mating season. In this species, there is a female preference for males with high throat UV reflectance, and males with high UV reflectance are more likely to win fights. We found that (i) while food shortage decreased lizards'' body condition, it did not affect colour development, and (ii) the available time for maintaining high body temperature affected the development of UV colour without affecting body condition or other colour traits.

Conclusions/Significance

Our results demonstrate that the expression of a sexually selected structural colour signal depends on the time at elevated body temperature affecting physiological performance but not on available energy gained from food per se in an ectothermic vertebrate. We suggest that the effect of high ambient temperature on UV colour in male L. viridis makes it an honest signal, because success in acquiring thermally favourable territories and/or effective behavioural thermoregulation can both be linked to individual quality.     

Modern Lineages of Mycobacterium tuberculosis Exhibit Lineage-Specific Patterns of Growth and Cytokine Induction in Human Monocyte-Derived Macrophages

Background

Strains of Mycobacterium tuberculosis vary in virulence. Strains that have caused outbreaks in the United States and United Kingdom have been shown to subvert the innate immune response as a potential immune evasion mechanism. There is, however, little information available as to whether these patterns of immune subversion are features of individual strains or characteristic of broad clonal lineages of M. tuberculosis.

Methods

Strains from two major modern lineages (lineage 2 [East-Asian] and lineage 4 [Euro-American]) circulating in the Western Cape in South Africa as well as a comparator modern lineage (lineage 3 [CAS/Delhi]) were identified. We assessed two virulence associated characteristics: mycobacterial growth (in liquid broth and monocyte derived macrophages) and early pro-inflammatory cytokine induction.

Results

In liquid culture, Lineage 4 strains grew more rapidly and reached higher plateau levels than other strains (lineage 4 vs. lineage 2 p = 0.0024; lineage 4 vs. lineage 3 p = 0.0005). Lineage 3 strains were characterized by low and early plateau levels, while lineage 2 strains showed an intermediate growth phenotype. In monocyte-derived macrophages, lineage 2 strains grew faster than lineage 3 strains (p<0.01) with lineage 4 strains having an intermediate phenotype. Lineage 2 strains induced the lowest levels of pro-inflammatory TNF and IL-12p40 as compared to other lineages (lineage 2: median TNF 362 pg/ml, IL-12p40 91 pg/ml; lineage 3: median TNF 1818 pg/ml, IL-12p40 123 pg/ml; lineage 4: median TNF 1207 pg/ml, IL-12p40 205 pg/ml;). In contrast, lineage 4 strains induced high levels of IL-12p40 and intermediate level of TNF. Lineage 3 strains induced high levels of TNF and intermediate levels of IL-12p40.

Conclusions

Strains of M. tuberculosis from the three major modern strain lineages possess distinct patterns of growth and cytokine induction. Rapid growth and immune subversion may be key characteristics to the success of these strains in different human populations.     

Photosystem II Function and Dynamics in Three Widely Used Arabidopsis thaliana Accessions

Columbia-0 (Col-0), Wassilewskija-4 (Ws-4), and Landsberg erecta-0 (Ler-0) are used as background lines for many public Arabidopsis mutant collections, and for investigation in laboratory conditions of plant processes, including photosynthesis and response to high-intensity light (HL). The photosystem II (PSII) complex is sensitive to HL and requires repair to sustain its function. PSII repair is a multistep process controlled by numerous factors, including protein phosphorylation and thylakoid membrane stacking. Here we have characterized the function and dynamics of PSII complex under growth-light and HL conditions. Ws-4 displayed 30% more thylakoid lipids per chlorophyll and 40% less chlorophyll per carotenoid than Col-0 and Ler-0. There were no large differences in thylakoid stacking, photoprotection and relative levels of photosynthetic complexes among the three accessions. An increased efficiency of PSII closure was found in Ws-4 following illumination with saturation flashes or continuous light. Phosphorylation of the PSII D1/D2 proteins was reduced by 50% in Ws-4 as compared to Col-0 and Ler-0. An increase in abundance of the responsible STN8 kinase in response to HL treatment was found in all three accessions, but Ws-4 displayed 50% lower levels than Col-0 and Ler-0. Despite this, the HL treatment caused in Ws-4 the lagest extent of PSII inactivation, disassembly, D1 protein degradation, and the largest decrease in the size of stacked thylakoids. The dilution of chlorophyll-protein complexes with additional lipids and carotenoids in Ws-4 may represent a mechanism to facilitate lateral protein traffic in the membrane, thus compensating for the lack of a full complement of STN8 kinase. Nevertheless, additional PSII damage occurs in Ws-4, which exceeds the D1 protein synthesis capacity, thus leading to enhanced photoinhibition. Our findings are valuable for selection of appropriate background line for PSII characterization in Arabidopsis mutants, and also provide the first insights into natural variation of PSII protein phosphorylation.     

The role of PET-CT in detecting unknown primary tumour in patients with cervical lymph node metastases

PET-CT examination was conducted with 440 patients treated at the Department of Head and Neck Surgery, National Institute of Oncology, Budapest, between January 1, 2006 and December 31, 2010. Out of them 77 patients were selected with whom no examination of any sort (physical, pan-endoscopy, or any of the conventional imaging techniques) succeeded in identifying the primary tumour. In each case the primary examination (aspiration cytology and histology) verified cervical metastases, most of them being squamous cell carcinoma. The significance of PET-CT was retrospectively evaluated in cases of unknown primary tumour with verified cervical metastases. We tested the sensitivity of PET-CT in detection of the primary malignant tumour, and possible distant metastases or a second primary in order to plan an optimal treatment schedule for the patient. Patients with whom the examinations specified in the treatment protocol (physical examination, pan-endoscopy, conventional imaging, biopsy) had failed to diagnose the primary tumour were referred to PET-CT. In each case 18F-FDG tracer was used. In 21/77 patients (27%), the PET-CT yielded unequivocal evidence for the primary tumour confirmed by histology, as well. With 10 others (13%), the precarious diagnoses by various imaging techniques were confirmed by the PET-CT. False positive findings with PET-CT that were not verified either by histology or control examination tests occurred but in 10 patients (13%). Concerning the primary tumour, false negative result was obtained only with 3 patients (4%). It should be noted that their retrospective evaluation proved diagnostic errors, the primary tumours were visible in all the scans. With 33 patients (43%) PET-CT furnished no additional information compared to the previous examinations. In 10 patients, asymptomatic distant metastases and in 3 patients synchronous tumours were diagnosed. We also acknowledge that the significance of PET-CT using 18F-FDG is unquestionable in the detection of unknown primary tumours. It is strongly recommended to re-include the detection of unknown primaries in the approved national indication list of PET-CT. (Note, until January 1, 2008 it had been included!) PET-CT is capable of detecting a primary tumour, after all unsuccessful diagnostic examinations till then, in 25-40% of the cases. One cannot disregard the role and significance of PET-CT in the detection of asymptomatic synchronous tumours, or distant metastases. These benefits make PET-CT a suitable tool for the refinement of individually tailored treatment strategies leading to better therapeutic results and more favourable cost-benefit ratio.