The prefusion structure of herpes simplex virus glycoprotein B.

Cell entry of enveloped viruses requires specialized viral proteins that mediate fusion with the host membrane by substantial structural rearrangements from a metastable pre- to a stable postfusion conformation. This metastability renders the herpes simplex virus 1 (HSV-1) fusion glycoprotein B (gB) highly unstable such that it readily converts into the postfusion form, thereby precluding structural elucidation of the pharmacologically relevant prefusion conformation. By identification of conserved sequence signatures and molecular dynamics simulations, we devised a mutation that stabilized this form. Functionally locking gB allowed the structural determination of its membrane-embedded prefusion conformation at sub-nanometer resolution and enabled the unambiguous fit of all ectodomains. The resulting pseudo-atomic model reveals a notable conservation of conformational domain rearrangements during fusion between HSV-1 gB and the vesicular stomatitis virus glycoprotein G, despite their very distant phylogeny. In combination with our comparative sequence-structure analysis, these findings suggest common fusogenic domain rearrangements in all class III viral fusion proteins.

Temporal decoding of vocal and musical emotions: Same code, different timecourse?

From a baby's cry to a piece of music, we perceive emotions from our auditory environment every day. Many theories bring forward the concept of common neural substrates for the perception of vocal and musical emotions. It has been proposed that, for us to perceive emotions, music recruits emotional circuits that evolved for the processing of biologically relevant vocalizations (e.g., screams, laughs). Although some studies have found similarities between voice and instrumental music in terms of acoustic cues and neural correlates, little is known about their processing timecourse. To further understand how vocal and instrumental emotional sounds are perceived, we used EEG to compare the neural processing timecourse of both stimuli type expressed with a varying degree of complexity (vocal/musical affect bursts and emotion-embedded speech/music). Vocal stimuli in general, as well as musical/vocal bursts, were associated with a more concise sensory trace at initial stages of analysis (smaller N1), although vocal bursts had shorter latencies than the musical ones. As for the P2 - vocal affect bursts and Emotion-Embedded Musical stimuli were associated with earlier P2s. These results support the idea that emotional vocal stimuli are differentiated early from other sources and provide insight into the common neurobiological underpinnings of auditory emotions.

Psychological intervention (ConquerFear) for treating fear of cancer recurrence: mediators and moderators of treatment efficacy.

PURPOSE: ConquerFear is an efficacious intervention for fear of cancer recurrence (FCR) that demonstrated greater improvements than an attention control (relaxation training) in a randomized controlled trial. This study aimed to determine mediators and moderators of the relative treatment efficacy of ConquerFear versus relaxation. METHODS: One hundred and fifty-two cancer survivors completed 5 therapy sessions and outcome measures before and after intervention and at 6 months' follow-up. We examined theoretically relevant variables as potential mediators and moderators of treatment outcome. We hypothesized that metacognitions and intrusions would moderate and mediate the relationship between treatment group and FCR level at follow-up. RESULTS: Only total FCR score at baseline moderated treatment outcome. Participants with higher levels of FCR benefited more from ConquerFear relative to relaxation on the primary outcome. Changes in metacognitions and intrusive thoughts about cancer during treatment partially mediated the relationship between treatment group and FCR. CONCLUSIONS: These results show that ConquerFear is relatively more effective than relaxation for those with overall higher levels of FCR. The mediation analyses confirmed that the most likely mechanism of treatment efficacy was the reduction in unhelpful metacognitions and intrusive thoughts during treatment, consistent with the theoretical framework underpinning ConquerFear. IMPLICATIONS FOR CANCER SURVIVORS: ConquerFear is a brief, effective treatment for FCR in cancer survivors with early-stage disease. The treatment works by reducing intrusive thoughts about cancer and changing beliefs about worry and is particularly helpful for people with moderate to severe FCR.

National assessment of ecosystem services in Russia: Methodology and main problems.

The Project TEEB-Russia is the first attempt at a nation-wide assessment of ecosystem services (ES) in Russia. The result of the pilot phase of the project (2013-2015) was the "Prototype National Report on Ecosystem Services of Russia, Volume 1, Terrestrial Ecosystems Services". A methodology for ES assessment was developed with allowance for the current status of the national public statistics. The units of ES assessment were the constituent entities of the Russian Federation. ES volumes supplied by ecosystems and consumed by humans were assessed. A number of productive (provisioning) and environment-forming (regulating) ES were evaluated in terms of quantitative scientific indicators; others were scored in terms of natural and socioeconomic factors that determine the supply and use of ES. The degree to which ES are consumed by humans was assessed by the ratio of supplied ES volumes to consumed ES volumes. The constituents of the Russian Federation were compared with respect to the balance of natural factors determining ES supply and socioeconomic factors determining ES use. Analyses of available data show that a national ES assessment can be started on the basis of the current national system of data collection. The creation of a full national system for assessment and monitoring of ES requires, in the short term, solving the problem of extremely unequal areas of administrative regions, ensuring availability and regular updating of data relevant for ES assessment that are already being collected by federal agencies as well as of the satellite maps of terrestrial ecosystems of Russia, and elaborating models linking ES volume to land cover types for the entire territory of Russia.

Microcarriers for high-pressure freezing and cryosectioning of adherent cells.

A method is described employing microcarrier spheres of cross-linked dextran for obtaining ultra- and semithin vitreous sections from high-pressure frozen anchorage-dependent (mammalian) cells. Avoiding trypsination or scraping cells off from the culture surface, the presented approach allows for cryoimmobilization, cryosectioning and cryoelectron microscopy/tomography of frozen-hydrated cells in an unperturbed manner which is important to preserve the native state of, for instance, the cytoskeleton. Furthermore, our studies on the 'life cycle' of Herpes simplex virus in Vero cells demonstrate that cell monolayers on microcarrier beads are well suited for fluorescence microscopic characterization of the sample prior to high-pressure freezing.

Insights into bunyavirus architecture from electron cryotomography of Uukuniemi virus.

Bunyaviridae is a large family of viruses that have gained attention as "emerging viruses" because many members cause serious disease in humans, with an increasing number of outbreaks. These negative-strand RNA viruses possess a membrane envelope covered by glycoproteins. The virions are pleiomorphic and thus have not been amenable to structural characterization using common techniques that involve averaging of electron microscopic images. Here, we determined the three-dimensional structure of a member of the Bunyaviridae family by using electron cryotomography. The genome, incorporated as a complex with the nucleoprotein inside the virions, was seen as a thread-like structure partially interacting with the viral membrane. Although no ordered nucleocapsid was observed, lateral interactions between the two membrane glycoproteins determine the structure of the viral particles. In the most regular particles, the glycoprotein protrusions, or "spikes," were seen to be arranged on an icosahedral lattice, with T = 12 triangulation. This arrangement has not yet been proven for a virus. Two distinctly different spike conformations were observed, which were shown to depend on pH. This finding is reminiscent of the fusion proteins of alpha-, flavi-, and influenza viruses, in which conformational changes occur in the low pH of the endosome to facilitate fusion of the viral and host membrane during viral entry.

Implementation and evaluation of a new workflow for registration and segmentation of pulmonary MRI data for regional lung perfusion assessment.

Recently it has been shown that regional lung perfusion can be assessed using time-resolved contrast-enhanced magnetic resonance (MR) imaging. Quantification of the perfusion images has been attempted, based on definition of small regions of interest (ROIs). Use of complete lung segmentations instead of ROIs could possibly increase quantification accuracy. Due to the low signal-to-noise ratio, automatic segmentation algorithms cannot be applied. On the other hand, manual segmentation of the lung tissue is very time consuming and can become inaccurate, as the borders of the lung to adjacent tissues are not always clearly visible. We propose a new workflow for semi-automatic segmentation of the lung from additionally acquired morphological HASTE MR images. First the lung is delineated semi-automatically in the HASTE image. Next the HASTE image is automatically registered with the perfusion images. Finally, the transformation resulting from the registration is used to align the lung segmentation from the morphological dataset with the perfusion images. We evaluated rigid, affine and locally elastic transformations, suitable optimizers and different implementations of mutual information (MI) metrics to determine the best possible registration algorithm. We located the shortcomings of the registration procedure and under which conditions automatic registration will succeed or fail. Segmentation results were evaluated using overlap and distance measures. Integration of the new workflow reduces the time needed for post-processing of the data, simplifies the perfusion quantification and reduces interobserver variability in the segmentation process. In addition, the matched morphological data set can be used to identify morphologic changes as the source for the perfusion abnormalities.

Sensitive detection of tumour cells in effusions by combining cytology and fluorescence in situ hybridisation (FISH).

Diagnosis of malignant cells in effusions is important for staging procedures and resulting therapeutic decisions. Cytodiagnostics in effusions is sometimes difficult since reactive mesothelial cells can mimic malignant cells. We used fluorescence in situ hybridisation (FISH) in single-colour or if appropriate in dual-colour evaluation to detect chromosomal aberrations in effusion cells as markers of malignancy, to raise the diagnostic yield. Cytologic and FISH evaluations--by using probes representing several chromosomes always including chromosomes 11 and 17--were performed in 358 effusion fluids. Cytology was positive for malignancy in 44.4% of all effusions, whereas FISH was positive in 53.9% (P=0.0001). The combination of cytology and FISH was diagnostic for malignancy in 60.9% of effusions. Diagnostic superiority of FISH was demonstrated in effusions from breast cancer, lung cancer, pancreatic cancer, and in effusions from the entire group of gynaecological and gastrointestinal carcinomas. In transudates (effusion protein <2.5 g dl(-1)), malignant cells were detectable by cytology, FISH, and combined use of both methods in 18.6, 30, and 37.1% of effusions, respectively, suggesting that cytologic and molecular analysis should be performed also with transudates. In conclusion, FISH in combination with conventional cytology is a highly sensitive and specific diagnostic tool for detecting malignant cells in effusions.

Association of NQO1 polymorphism with spontaneous breast cancer in two independent populations.

Eight different single-nucleotide polymorphisms (SNPs) in six different genes were investigated for possible association with breast cancer. We used a case-control study design in two Caucasian populations, one from Tyrol, Austria, and the other from Prague, Czech Republic. Two SNPs showed an association with breast cancer: R72P inTP53 and P187S in NQO1. Six SNPs, Q356R and P871L in BRCA1, N372H in BRCA2, C112R (E4) and R158C (E2) in ApoE and C825T in GNB3, did not show any sign of association. The P187S polymorphism in NQO1 was associated with breast cancer in both populations from Tyrol and Prague with a higher risk for carriers of the 187S allele. Combining the results of the two populations, we observed a highly significant difference (P=0.0004) of genotype and allele frequencies (odds ratio (OR)=1.46; 95% confidence interval (CI) 1.16-1.85; P=0.001) and of the homozygote ratio (OR=3.8; 95% CI 1.73-8.34; P=0.0001). Combining the two 'candidate' SNPs (P187S and R72P) revealed an increased risk for breast cancer of double heterozygotes (P187S/R72P) of the NQO1 and TP53 genes (OR=1.88; 95% CI 1.13-3.15; P=0.011), suggesting a possible interaction of these two loci.

Spontaneous remission in a secondary acute myelogenous leukaemia following invasive pulmonary aspergillosis.

Spontaneous remission of adult acute myelogenous leukaemia (AML) represents a rare event. We report a 60-year-old female patient suffering from secondary AML M1 and severe invasive pulmonary hyalohyphomycosis highly suggestive of aspergillosis. Two months after the diagnosis of leukaemia, she achieved a spontaneous remission lasting 3 months, although neither cytostatic drugs nor corticoids were administered because of a septic condition. At the time of remission, a chronic hepatitis C virus infection and a polyclonal hypergammaglobulinaemia were present, and the patient received granulocyte colony-stimulating factor once. This report represents the first documentation of a spontaneous remission in AML following invasive pulmonary hyalohyphomycosis. Possible mechanisms of this phenomenon are discussed.

Ketocarotenoid biosynthesis outside of plastids in the unicellular green alga Haematococcus pluvialis.

The carotenoid biosynthetic pathway in algae and plants takes place within plastids. In these organelles, carotenoids occur either in a free form or bound to proteins. Under stress, the unicellular green alga Haematococcus pluvialis accumulates secondary carotenoids, mainly astaxanthin esters, in cytoplasmic lipid vesicles up to 4% of its dry mass. It is therefore one of the favored organisms for the biotechnological production of these antioxidative compounds. We have studied the cellular localization and regulation of the enzyme beta-carotene oxygenase in H. pluvialis that catalyzes the introduction of keto functions at position C-4 of the beta-ionone ring of beta-carotene and zeaxanthin. Using immunogold labeling of ultrathin sections and Western blot analysis of cell fractions, we discovered that under inductive conditions, beta-carotene oxygenase was localized both in the chloroplast and in the cytoplasmic lipid vesicles, which are (according to their lipid composition) derived from cytoplasmic membranes. However, beta-carotene oxygenase activity was confined to the lipid vesicle compartment. Because an early carotenogenic enzyme in the pathway, phytoene desaturase, was found only in the chloroplast (Grünewald, K., Eckert, M., Hirschberg, J., and Hagen, C. (2000) Plant Physiol. 122, 1261-1268), a transport of intermediates from the site of early biosynthetic steps in the chloroplast to the site of oxygenation and accumulation in cytoplasmic lipid vesicles is proposed.

Evidence from a leukaemia model for maintenance of vascular endothelium by bone-marrow-derived endothelial cells.

BACKGROUND: Vascular endothelial cells lost from the blood-vessel endothelium through necrosis or apoptosis must be replaced. We investigated in a leukaemia model whether bone-marrow-derived endothelial cells contribute to this maintenance angiogenesis. METHODS: We studied six patients with chronic myelogenous leukaemia (CML) carrying the BCR/ABL fusion gene in their bone-marrow-derived cells. We screened endothelial cells generated in vitro from bone-marrow-derived progenitor cells and vascular endothelium in myocardial tissue for the BCR/ABL fusion gene by in-situ hybridisation. For detection of donor-type endothelial cells after transplantation of haemopoietic stem cells, recipient tissue was stained with monoclonal antibodies against donor-type HLA antigens. FINDINGS: We identified the BCR/ABL fusion gene in variable proportions (0-56%) of endothelial cells generated in vitro. Endothelial cells expressing the fusion gene were found in the vascular endothelium of a patient. In a recipient of an allogeneic stem-cell transplant, normal donor-type endothelial cells were detected in the vascular endothelium. INTERPRETATION: These findings suggest that CML is not solely a haematological disease but originates from a bone-marrow-derived haemangioblastic precursor cell that can give rise to both blood cells and endothelial cells. Moreover, normal bone-marrow-derived endothelial cells can contribute to the maintenance of the blood vascular endothelium. The integration of bone-marrow-derived endothelial cells into the vascular endothelium provides a rationale for developing vascular targeting strategies in vasculopathies, inflammatory diseases, and cancer.

Mammaglobin gene expression: a superior marker of breast cancer cells in peripheral blood in comparison to epidermal-growth-factor receptor and cytokeratin-19.

Various molecular markers have been used for the detection of circulating breast cancer cells in blood by reverse transcriptase-polymerase chain reaction (RT-PCR). Using nested RT-PCR, we compared the specificity and sensitivity of human mammaglobin (hMAM), epidermal-growth-factor receptor (EGF-R), and cytokeratin 19 (CK-19) expression as markers for circulating carcinoma cells in the peripheral blood of patients with breast cancer. Blood samples from 12 patients with ductal carcinoma in situ, 133 patients with invasive breast cancer, 20 patients with hematological malignancies, 31 healthy volunteers, and tumor tissues from 40 patients with invasive breast cancer were screened for mRNA encoding hMAM, EGF-R, or CK-19 by nested RT-PCR. In all breast cancer tissues, mRNA for hMAM, EGF-R, and CK-19 was detectable. In blood samples from patients with invasive breast cancer, 11 (8%), 13 (10%), and 64 (48%) were positive for mRNA encoding hMAM, EGF-R, or CK-19, respectively. Blood samples from none of the healthy volunteers and patients with hematological disorders were positive for hMAM, while CK-19 mRNA was found in the blood of 12 (39%) healthy volunteers and transcripts for EGF-R and CK-19 were detectable in 5 (25%) and 2 (10%), respectively, of the patients with hematological malignancies. Only hMAM mRNA expression in blood correlated with clinical parameters such as nodal status, metastasis, and CA 15-3 serum levels. In summary, hMAM transcripts detectable in blood by RT-PCR represent the most specific molecular marker for hematogenous spread of breast cancer cells. With the nested RT-PCR method, aberrant EGF-R mRNA expression might occasionally be found in hematological malignancies, whereas CK-19 mRNA expression proved to be rather nonspecific. The prognostic value of hMAM RT-PCR-based tumor cell detection in peripheral blood should be further tested and validated in prospective studies.

Dietary protein does not affect overloaded skeletal muscle in rats.

We compared the effects of three levels of dietary protein, i.e., 7% (low protein; LP); 17.5% (adequate protein; CON); or 30% (high protein; HP) on growth of functionally overloaded muscle in Sprague-Dawley male rats. Growth of plantaris and soleus muscles was induced by the surgical removal of gastrocnemius muscles in one hindlimb; muscles in the other leg were used as sham-operated, intra-animal controls. After 4 wk, rats fed the 7% LP diet gained less weight (-29%) and had lighter livers (-20%) and kidneys (-16%) than rats fed the CON diet (P < 0.05). Measurements of rats fed the 30% HP diet were not different from those of CON rats except that their kidneys were larger (+6%) (P < 0.05). The level of dietary protein did not affect the experimentally induced muscular growth in either plantaris or soleus muscles. Gains in overloaded plantaris muscles over sham-operated muscles were not different among rats fed LP, CON and HP diets for muscle mass (+42 to +45%), total protein (+42 to +46%) and myofibrillar protein (+40 to +44%). Soleus muscles also did not differ among diet groups for gains in mass (+20 to +33%), total protein (+20 to +33%) and myofibrillar protein (+21 to +33%). No dietary protein effects were found on myosin heavy chain isoform (I, IIa, IIx, IIb) expression in either plantaris or soleus muscles. We conclude that gains in plantaris and soleus muscle mass, total protein and myofibrillar protein induced by functional overload are not affected by low (7%) or high (30%) protein feeding in young male rats for 4 wk.

New rearrangement pattern after treatment of hairy-cell leukemia with 2-chlorodeoxyadenosine.

Leukemic hairy cells are clonally proliferating B-lymphoid cells with clonal rearrangements of genes for immunoglobulin chains. We describe a patient with a new hairy-cell clone after treatment with 2-chlorodeoxyadenosine (2-CdA). In this patient, a single course of 2-CdA resulted in good partial remission of hairy-cell leukemia, but Southern blot analysis of bone marrow biopsies and polymerase chain reaction using seminested amplifications with consensus primers revealed a new rearranged band 4 months after therapy with 2-CdA. Four years after therapy, the patient is in complete clinical remission and both bands disappeared during follow-up. The new rearranged band might have been related to prior treatment of hairy-cell leukemia with 2-CdA.

Phytoene desaturase is localized exclusively in the chloroplast and up-regulated at the mRNA level during accumulation of secondary carotenoids in Haematococcus pluvialis (Volvocales, chlorophyceae).

The unicellular green alga Haematococcus pluvialis Flotow is known for its massive accumulation of ketocarotenoids under various stress conditions. Therefore, this microalga is one of the favored organisms for biotechnological production of these antioxidative compounds. Astaxanthin makes up the main part of the secondary carotenoids and is accumulated mostly in an esterified form in extraplastidic lipid vesicles. We have studied phytoene desaturase, an early enzyme of the carotenoid biosynthetic pathway. The increase in the phytoene desaturase protein levels that occurs following induction is accompanied by a corresponding increase of its mRNA during the accumulation period, indicating that phytoene desaturase is regulated at the mRNA level. We also investigated the localization of the enzyme by western-blot analysis of cell fractions and by immunogold labeling of ultrathin sections for electron microscopy. In spite of the fact that secondary carotenoids accumulate outside the chloroplast, no extra pathway specific for secondary carotenoid biosynthesis in H. pluvialis was found, at least at this early stage in the biosynthesis. A transport process of carotenoids from the site of biosynthesis (chloroplast) to the site of accumulation (cytoplasmatic located lipid vesicles) is implicated.

The effects of dietary energy restriction on overloaded skeletal muscle in rats.

We evaluated the effects of three levels of energy intake, 73 % (CON73), 81 % (CON81) and 100 % (CON100) of the ad libitum intake of the control diet, on skeletal muscle growth induced by functional overload in male rats. Unlike most previous studies which have employed chronic or acute food restriction where all nutrients are reduced in the diet, the present study tested the effects of energy deprivation as a single factor without inducing other nutritional deficiencies. Muscular growth of plantaris and soleus muscles was induced by removal of synergist gastrocnemius muscles in one hindlimb; muscles in the other leg were used as sham-operated intra-animal controls. After 30 d, rats on the energy-restricted CON73 and CON81 diets gained less weight and had smaller livers, kidneys, hearts and fat pads (epididymal, retroperitoneal and omental) than CON100 rats They also had smaller sham-operated plantaris muscles (CON73 --13 %, CON81 --9 %) containing less total protein (CON73 --14 %; CON81 --10 %) than CON100 rats However, the same measurements in overloaded plantaris muscles were similar among groups. Soleus muscle mass and protein contents were not significantly affected by energy restriction in our study. Percentage distributions of myosin heavy-chain isoforms (types I, IIa, IIx and IIb) were similar among rats in CON100, CON81 and CON73 groups for both plantaris and soleus muscles. We conclude that the growth reduction of plantaris muscle induced by energy restriction at 73 % and 81 % for 30 d was prevented by functional overload in male rats.

Surgical treatment of interventional coronary angiographic accidents.

Newer methodologies have increased the incidence of coronary interventions. At the authors' institution, 5,614 coronary interventional procedures (28% of all catheterizations) were performed over a 3-year period, from 1995 to 1997. Eighty-one patients (1.4%) suffered angiographic accidents, including coronary artery dissection, free rupture, tamponade, foreign body embolism, and wire entrapment, and were retrospectively reviewed. All patients were taken for emergency surgery in less than 4 hours. The mean age was 61.2 years, 44 (54%) were men, and 37 (46%) were in cardiogenic shock at the time of surgery. Fifty-seven patients (70%) had intraaortic balloon counterpulsation. The number of previous cardiac interventions ranged from one to four with a mean of 1.9. One to five bypass grafts (mean, 2.2) were performed, and three patients required temporary ventricular assist devices. There were six deaths for a 30-day mortality rate of 7.4%. Thirty-two patients (39.5%) suffered significant morbidity, including cerebrovascular accidents, and renal and respiratory failure. Perioperative myocardial infarctions were diagnosed in 39 (48%) patients. Average length of stay was 12.1 days. One-year survival was satisfactory at 90% (73/81), with 56 survivors (77%) regaining normal everyday activity. Early surgical intervention, rapid revascularization, and temporary mechanical support are keys to low mortality in this high-risk group. Identification of high-risk interventions and significant comorbid conditions, with concomitant surgical consultation, need to be pursued to reduce the high morbidity rate.