Elucidation of an archaeal replication protein network to generate enhanced PCR enzymes.

Thermostable DNA polymerases are an important tool in molecular biology. To exploit the archaeal repertoire of proteins involved in DNA replication for use in PCR, we elucidated the network of proteins implicated in this process in Archaeoglobus fulgidus. To this end, we performed extensive yeast two-hybrid screens using putative archaeal replication factors as starting points. This approach yielded a protein network involving 30 proteins potentially implicated in archaeal DNA replication including several novel factors. Based on these results, we were able to improve PCR reactions catalyzed by archaeal DNA polymerases by supplementing the reaction with predicted polymerase co-factors. In this approach we concentrated on the archaeal proliferating cell nuclear antigen (PCNA) homologue. This protein is known to encircle DNA as a ring in eukaryotes, tethering other proteins to DNA. Indeed, addition of A. fulgidus PCNA resulted in marked stimulation of PCR product generation. The PCNA-binding domain was determined, and a hybrid DNA polymerase was constructed by grafting this domain onto the classical PCR enzyme from Thermus aquaticus, Taq DNA polymerase. Addition of PCNA to PCR reactions catalyzed by the fusion protein greatly stimulated product generation, most likely by tethering the enzyme to DNA. This sliding clamp-induced increase of PCR performance implies a promising novel micromechanical principle for the development of PCR enzymes with enhanced processivity.     

“Into and Out of” the Qinghai‐Tibet Plateau and the Himalayas: Centers of origin and diversification across five clades of Eurasian montane and alpine passerine birds

Encompassing some of the major hotspots of biodiversity on Earth, large mountain systems have long held the attention of evolutionary biologists. The region of the Qinghai‐Tibet Plateau (QTP) is considered a biogeographic source for multiple colonization events into adjacent areas including the northern Palearctic. The faunal exchange between the QTP and adjacent regions could thus represent a one‐way street (“out of” the QTP). However, immigration into the QTP region has so far received only little attention, despite its potential to shape faunal and floral communities of the QTP. In this study, we investigated centers of origin and dispersal routes between the QTP, its forested margins and adjacent regions for five clades of alpine and montane birds of the passerine superfamily Passeroidea. We performed an ancestral area reconstruction using BioGeoBEARS and inferred a time‐calibrated backbone phylogeny for 279 taxa of Passeroidea. The oldest endemic species of the QTP was dated to the early Miocene (ca. 20 Ma). Several additional QTP endemics evolved in the mid to late Miocene (12–7 Ma). The inferred centers of origin and diversification for some of our target clades matched the “out of Tibet hypothesis’ or the “out of Himalayas hypothesis” for others they matched the “into Tibet hypothesis.” Three radiations included multiple independent Pleistocene colonization events to regions as distant as the Western Palearctic and the Nearctic. We conclude that faunal exchange between the QTP and adjacent regions was bidirectional through time, and the QTP region has thus harbored both centers of diversification and centers of immigration.     

Anaerobic Ammonia Oxidation in the Presence of Nitrogen Oxides (NOx) by Two Different Lithotrophs

The anaerobic ammonia-oxidizing activity of the planctomycete Candidatus “Brocadia anammoxidans” was not inhibited by NO concentrations up to 600 ppm and NO₂ concentrations up to 100 ppm. B. anammoxidans was able to convert (detoxify) NO, which might explain the high NO tolerance of this organism. In the presence of NO₂, the specific ammonia oxidation activity of B. anammoxidans increased, and Nitrosomonas-like microorganisms recovered an NO₂-dependent anaerobic ammonia oxidation activity. Addition of NO₂ to a mixed population of B. anammoxidans and Nitrosomonas induced simultaneous specific anaerobic ammonia oxidation activities of up to 5.5 mmol of NH₄⁺ g of protein⁻¹ h⁻¹ by B. anammoxidans and up to 1.5 mmol of NH₄⁺ g of protein⁻¹ h⁻¹ by Nitrosomonas. The stoichiometry of the converted N compounds (NO₂⁻/NH₃ ratio) and the microbial community structure were strongly influenced by NO₂. The combined activity of B. anammoxidans and Nitrosomonas-like ammonia oxidizers might be of relevance in natural environments and for technical applications.     

Anaerobic ammonia oxidation with nitrogen dioxide by Nitrosomonas eutropha

Nitrosomonas eutropha, an obligately lithoautotrophic bacterium, was able to nitrify and denitrify simultaneously under anoxic conditions when gaseous nitrogen dioxide (NO₂) was supplemented to the atmosphere. In the presence of gaseous NO₂, ammonia was oxidized, nitrite and nitric oxide (NO) were formed, and hydroxylamine occurred as an intermediate. Between 40 and 60% of the produced nitrite was denitrified to dinitrogen (N₂). Nitrous oxide (N₂O) was shown to be an intermediate of denitrification. Under an N₂ atmosphere supplemented with 25 ppm NO₂ and 300 ppm CO₂, the amount of cell protein increased by 0.87 mg protein per mmol ammonia oxidized, and the cell number of N. eutropha increased by 5.8 × 10⁹ cells per mmol ammonia oxidized. In addition, the ATP and NADH content increased by 4.3 μmol ATP (g protein)^–1 and 6.3 μmol NADH (g protein)^–1 and was about the same in both anaerobically and aerobically grown cells. Without NO₂, the ATP content decreased by 0.7 μmol (g protein)^–1, and the NADH content decreased by 1.2 μmol (g protein)^–1. NO was shown to inhibit anaerobic ammonia oxidation. Received: 9 October 1996 / Accepted: 5 December 1996     

EST sequencing of Onychophora and phylogenomic analysis of Metazoa

Onychophora (velvet worms) represent a small animal taxon considered to be related to Euarthropoda. We have obtained 1873 5' cDNA sequences (expressed sequence tags, ESTs) from the velvet worm Epiperipatus sp., which were assembled into 833 contigs. BLAST similarity searches revealed that 51.9% of the contigs had matches in the protein databases with expectation values lower than 10(-4). Most ESTs had the best hit with proteins from either Chordata or Arthropoda (approximately 40% respectively). The ESTs included sequences of 27 ribosomal proteins. The orthologous sequences from 28 other species of a broad range of phyla were obtained from the databases, including other EST projects. A concatenated amino acid alignment comprising 5021 positions was constructed, which covers 4259 positions when problematic regions were removed. Bayesian and maximum likelihood methods place Epiperipatus within the monophyletic Ecdysozoa (Onychophora, Arthropoda, Tardigrada and Nematoda), but its exact relation to the Euarthropoda remained unresolved. The "Articulata" concept was not supported. Tardigrada and Nematoda formed a well-supported monophylum, suggesting that Tardigrada are actually Cycloneuralia. In agreement with previous studies, we have demonstrated that random sequencing of cDNAs results in sequence information suitable for phylogenomic approaches to resolve metazoan relationships.     

Modified Vaccinia Virus Ankara Exerts Potent Immune Modulatory Activities in a Murine Model

Background

Modified vaccinia virus Ankara (MVA), a highly attenuated strain of vaccinia virus, has been used as vaccine delivery vector in preclinical and clinical studies against infectious diseases and malignancies. Here, we investigated whether an MVA which does not encode any antigen (Ag) could be exploited as adjuvant per se.

Methodology/Principal Findings

We showed that dendritic cells infected in vitro with non-recombinant (nr) MVA expressed maturation and activation markers and were able to efficiently present exogenously pulsed Ag to T cells. In contrast to the dominant T helper (Th) 1 biased responses elicited against Ags produced by recombinant MVA vectors, the use of nrMVA as adjuvant for the co-administered soluble Ags resulted in a long lasting mixed Th1/Th2 responses.

Conclusions/Significance

These findings open new ways to potentiate and modulate the immune responses to vaccine Ags depending on whether they are co-administered with MVA or encoded by recombinant viruses.     

Jasmonic acid perception by COI1 involves inositol polyphosphates in Arabidopsis thaliana

Plant responses to wounding are part of their defense responses against insects, and are tightly regulated. The isoleucin conjugate of jasmonic acid (JA‐Ile) is a major regulatory molecule. We have previously shown that inositol polyphosphate signals are required for defense responses in Arabidopsis; however, the way in which inositol polyphosphates contribute to plant responses to wounding has so far remained unclear. Arabidopsis F‐box proteins involved in the perception of JA‐Ile (COI1) and auxin (TIR1) are structurally similar. Because TIR1 has recently been shown to contain inositol hexakisphosphate (InsP₆) as a co‐factor of unknown function, here we explored the possibility that InsP₆ or another inositol polyphosphate is required for COI1 function. In support of this hypothesis, COI1 variants with changes in putative inositol polyphosphate coordinating residues exhibited a reduced interaction with the COI1 target, JAZ9, in yeast two‐hybrid tests. The equivalent COI1 variants displayed a reduced capability to rescue jasmonate‐mediated root growth inhibition or silique development in Arabidopsis coi1 mutants. Yeast two‐hybrid tests using wild‐type COI1 in an ipk1Δ yeast strain exhibiting increased levels of inositol pentakisphosphate (InsP₅) and reduced levels of InsP₆ indicate an enhanced COI1/JAZ9 interaction. Consistent with these findings, Arabidopsis ipk1‐1 mutants, also with increased InsP₅ and reduced InsP₆ levels, showed increased defensive capabilities via COI1‐mediated processes, including wound‐induced gene expression, defense against caterpillars or root growth inhibition by jasmonate. The combined data from experiments using mutated COI1 variants, as well as yeast and Arabidopsis backgrounds altered in inositol polyphosphate metabolism, indicate that an inositol polyphosphate, and probably InsP₅, contributes to COI1 function.     

Low-field magnetic resonance imaging study on carpal arthritis in systemic sclerosis - low-grade erosive arthritis of carpal bones is an unexpected and frequent disease manifestation

Introduction

The aim of the present study was to assess the prevalence and characteristics of subclinical arthritis of carpal and metacarpophalangeal joints in patients with systemic sclerosis (SSc).

Methods

Low-field (0.2 T) magnetic resonance imaging (MRI) was performed in consecutive patients with SSc attending our center between January 2010 and March 2011. Results were assessed in a standardized manner using the Rheumatoid Arthritis Magnetic Resonance Imaging Score (RAMRIS) and standardized assessments of all hand joints. Patients with arthritis due to overlap syndromes were excluded.

Results

Of 38 inpatients and eight outpatients who were screened for inclusion, 30 patients participated in the study and 26 patients could be evaluated. Erosions, bone marrow edema, synovitis, and joint effusions were found in 87%, 37%, 68%, and 58%, respectively, and 24% of patients had additional tenovaginitis. Arthritis affected only a low number of joints per analyzed hand. All bones and joints could be affected, but synovitis and bone marrow edema occurred predominantly in the proximal row of carpal bones, most frequently affecting the lunate bone. The extent of inflammatory changes measured with the RAMRIS correlated significantly with the functional status assessed with the validated German functional score questionnaire Funktionsfragebogen Hannover.

Conclusion

Low-grade arthritic changes on low-field MRI are frequent in patients with pure SSc. The features of arthritis in SSc differ from rheumatoid arthritis. The distribution, the MRI pattern and the predilection for the lunate bone raise the hypothesis that arthritis in SSc may be caused not only by immunological inflammation but also by ischemic mechanisms.     

Behavioural Lateralization in Budgerigars Varies with the Task and the Individual

Handedness/footedness and side biases are a well-known phenomenon in many animals, including humans. However, these so-called biases have mostly been studied at the population level - individual biases have received less attention, especially with regard to consistency over different tasks. Here we investigate behavioral lateralization in 12 male Budgerigars, Melopsittacus undulatus, a social parrot inhabiting the Australian bushlands. We performed 5 types of experiments to investigate lateralization, in tasks that involved climbing onto a perch, or landing on perches arranged in various configurations. The birds displayed highly significant, individually varying biases. The bias displayed by any particular individual varied with the task, in strength as well as polarity. Analysis of the data revealed that the preferred foot used for climbing did not coincide with the foot that was used while landing. Thus, landing choices are probably not determined by foot bias. Furthermore, these individual preferences were overridden completely when a bird had to perform a task simultaneously with another bird.     

Diffusion- and Perfusion-Weighted Imaging in Acute Lacunar Infarction: Is There a Mismatch?

Purpose

Characterization of lacunar infarction (LI) by use of multimodal MRI including diffusion- and perfusion-weighted imaging (DWI, PWI) is difficult because of the small lesion size. Only a few studies evaluated PWI in LI and the results are inconsistent.

Methods

In 16 LI patients who underwent initial MRI within 6 hours after symptom onset and follow-up MRI within 1 week demographics, clinical presentation, and MRI findings were analyzed with special emphasis on DWI and PWI findings. Time to peak maps were classified as showing a normal perfusion pattern or areas of hypoperfusion which were further categorized in mismatch (PWI>DWI), inverse mismatch (PWI<DWI), and match (PWI=DWI). Quantitative perfusion maps were generated and analyzed by use of Signal Processing in NMR-Software (SPIN).

Results

Of the 16 patients (mean age 65.5±12.9 years), 14 (87.5%) were male. Clinical symptoms comprised dysarthria (50%), hemiparesis (81.3%), and hemihypaesthesia (18.8%). Intravenous thrombolysis was performed in 7 (43.8%) patients. Clinical improvement was observed in 12 patients (75 %), while 2 (12.5%) patients showed a deterioration and another 2 (12.5%) a stable course. Acute ischemic lesions (mean volume of 0.46±0.29 cm³) were located in the thalamus (n=8, 50%), internal capsule (n=4, 25%), corona Radiata (n=3, 18.8%) and the mesencephalon (n=1, 6.3%). Circumscribed hypoperfusion (mean volume 0.61±0.48 cm³) was evident in 10 (62.5%) patients. Of these, 3 patients demonstrated a match, 4 an inverse mismatch, and 3 a mismatch between DWI and PWI lesion. Mean CBF and CBV ratios were 0.65±0.28 and 0.84±0.41 respectively. Growth of DWI lesions was observed in 7 (43.8%) and reversal of DWI lesions in 3 (18.8%) patients.

Conclusions

MRI allows identification of different DWI and PWI patterns in LI, including growth and reversal of ischemic lesions. Consequently, it may serve for a better characterization of this stroke subtype and support treatment decisions in daily clinical practice.