Competitive fitness in coronaviruses is not correlated with size or number of double-membrane vesicles under reduced-temperature growth conditions.

Positive-stranded viruses synthesize their RNA in membrane-bound organelles, but it is not clear how this benefits the virus or the host. For coronaviruses, these organelles take the form of double-membrane vesicles (DMVs) interconnected by a convoluted membrane network. We used electron microscopy to identify murine coronaviruses with mutations in nsp3 and nsp14 that replicated normally while producing only half the normal amount of DMVs under low-temperature growth conditions. Viruses with mutations in nsp5 and nsp16 produced small DMVs but also replicated normally. Quantitative reverse transcriptase PCR (RT-PCR) confirmed that the most strongly affected of these, the nsp3 mutant, produced more viral RNA than wild-type virus. Competitive growth assays were carried out in both continuous and primary cells to better understand the contribution of DMVs to viral fitness. Surprisingly, several viruses that produced fewer or smaller DMVs showed a higher fitness than wild-type virus at the reduced temperature, suggesting that larger and more numerous DMVs do not necessarily confer a competitive advantage in primary or continuous cell culture. For the first time, this directly demonstrates that replication and organelle formation may be, at least in part, studied separately during infection with positive-stranded RNA virus. IMPORTANCE The viruses that cause severe acute respiratory syndrome (SARS), poliomyelitis, and hepatitis C all replicate in double-membrane vesicles (DMVs). The big question about DMVs is why they exist in the first place. In this study, we looked at thousands of infected cells and identified two coronavirus mutants that made half as many organelles as normal and two others that made typical numbers but smaller organelles. Despite differences in DMV size and number, all four mutants replicated as efficiently as wild-type virus. To better understand the relative importance of replicative organelles, we carried out competitive fitness experiments. None of these viruses was found to be significantly less fit than wild-type, and two were actually fitter in tests in two kinds of cells. This suggests that viruses have evolved to have tremendous plasticity in the ability to form membrane-associated replication complexes and that large and numerous DMVs are not exclusively associated with efficient coronavirus replication.

Management effects on colostrogenesis in small ruminants: a review

Colostrum feeding in small ruminants is crucial during the first hours after birth due to the lack of Ig transfer during pregnancy via the placenta. In addition the immature immune system of the neonate is slow to produce its own Ig during the first weeks of life. Colostrogenesis, i.e. the transfer of Ig from blood into mammary secretions, starts several weeks prepartum. In goat plasma, immunoglobulin G (IgG) concentration decreases by around 38% from the third month of gestation until partum, which coincides with the dry period. Thus, management during the dry period is crucial for the course of colostrogenesis. The colostrum synthesis is determined by the nutrition during the prepartum period, but the transfer of Ig is obviously independent of nutritional influences. The administration of conjugated linoleic acid during the dry period to dairy goats causes a less pronounced decrease of blood plasma IgG concentration (6%) but it did not change colostral IgG levels. In cattle, IgG1 is transported from blood into colostrum by an IgG1 specific receptor located on the surface of alveolar epithelial cells during colostrogenesis, and this is most likely similar in small ruminants. Via inactivation of this receptor, the Ig transfer is downregulated by increasing prolactin (PRL) during lactogenesis. It was recently observed in goats treated with PGF2 alpha, in order to induce parturition, lower colostrum IgG concentrations occurred concomitantly with an earlier increase of plasma PRL as compared to untreated animals. The effect of litter size and number of lactations on colostral IgG concentration in small ruminants has not been made fully clear until now most likely due to the different breeds used in the published studies.

Creatine and neurotrophin-4/5 promote survival of nitric oxide synthase-expressing interneurons in striatal cultures

Nitric oxide (NO) mediates a variety of physiological functions in the central nervous system and acts as an important developmental regulator. Striatal interneurons expressing neuronal nitric oxide synthase (nNOS) have been described to be relatively spared from the progressive cell loss in Huntington's disease (HD). We have recently shown that creatine, which supports the phosphagen energy system, induces the differentiation of GABAergic cells in cultured striatal tissue. Moreover, neurotrophin-4/5 (NT-4/5) has been found to promote the survival and differentiation of cultured striatal neurons. In the present study, we assessed the effects of creatine and NT-4/5 on nNOS-immunoreactive (-ir) neurons of E14 rat ganglionic eminences grown for 1 week in culture. Chronic administration of creatine [5mM], NT-4/5 [10ng/ml], or a combination of both factors significantly increased numbers of nNOS-ir neurons. NT-4/5 exposure also robustly increased levels of nNOS protein. Interestingly, only NT-4/5 and combined treatment significantly increased general viability but no effects were seen for creatine supplementation alone. In addition, NT-4/5 and combined treatment resulted in a significant larger soma size and number of primary neurites of nNOS-ir neurons while creatine administration alone exerted no effects. Double-immunolabeling studies revealed that all nNOS-ir cells co-localized with GABA. In summary, our findings suggest that creatine and NT-4/5 affect differentiation and/or survival of striatal nNOS-ir GABAergic interneurons. These findings provide novel insights into the biology of developing striatal neurons and highlight the potential of both creatine and NT-4/5 as therapeutics for HD.

Sample sizes of studies on diagnostic accuracy: literature survey

OBJECTIVES: To determine sample sizes in studies on diagnostic accuracy and the proportion of studies that report calculations of sample size. DESIGN: Literature survey. DATA SOURCES: All issues of eight leading journals published in 2002. METHODS: Sample sizes, number of subgroup analyses, and how often studies reported calculations of sample size were extracted. RESULTS: 43 of 8999 articles were non-screening studies on diagnostic accuracy. The median sample size was 118 (interquartile range 71-350) and the median prevalence of the target condition was 43% (27-61%). The median number of patients with the target condition--needed to calculate a test's sensitivity--was 49 (28-91). The median number of patients without the target condition--needed to determine a test's specificity--was 76 (27-209). Two of the 43 studies (5%) reported a priori calculations of sample size. Twenty articles (47%) reported results for patient subgroups. The number of subgroups ranged from two to 19 (median four). No studies reported that sample size was calculated on the basis of preplanned analyses of subgroups. CONCLUSION: Few studies on diagnostic accuracy report considerations of sample size. The number of participants in most studies on diagnostic accuracy is probably too small to analyse variability of measures of accuracy across patient subgroups.

Clinical signs of deformed wing virus infection are predictive markers for honey bee colony losses.

The ectoparasitic mite Varroa destructor acting as a virus vector constitutes a central mechanism for losses of managed honey bee, Apis mellifera, colonies. This creates demand for an easy, accurate and cheap diagnostic tool to estimate the impact of viruliferous mites in the field. Here we evaluated whether the clinical signs of the ubiquitous and mite-transmitted deformed wing virus (DWV) can be predictive markers of winter losses. In fall and winter 2007/2008, A.m. carnica workers with apparent wing deformities were counted daily in traps installed on 29 queenright colonies. The data show that colonies which later died had a significantly higher proportion of workers with wing deformities than did those which survived. There was a significant positive correlation between V. destructor infestation levels and the number of workers displaying DWV clinical signs, further supporting the mite's impact on virus infections at the colony level. A logistic regression model suggests that colony size, the number of workers with wing deformities and V. destructor infestation levels constitute predictive markers for winter colony losses in this order of importance and ease of evaluation.

Post mortem computed tomography and core needle biopsy in comparison to autopsy in eleven bernese mountain Dogs with histiocytic sarcoma

Background: Bernese mountain dogs are reported to have a shorter life expectancy than other breeds. A Major reason for this has been assigned to a high tumour prevalence, especially of histiocytic sarcoma. The efforts made by the breeding clubs to improve the longevity with the help of genetic tests and breeding value estimations are impeded by insufficiently reliable diagnoses regarding the cause of death. The current standard for post mortem examination in animals is performance of an autopsy. In human forensic medicine, imaging modalities, such as computed tomography and magnetic resonance imaging, are used with increasing frequency as a complement to autopsy. The present study investigates, whether post mortem computed tomography in combination with core needle biopsy is able to provide a definitive diagnosis of histiocytic sarcoma. For this purpose we have analysed the results of post mortem computed tomography and core needle biopsy in eleven Bernese mountain dogs. In the subsequent autopsy, every dog had a definitive diagnosis of histiocytic sarcoma, based on immunohistochemistry. Results: Computed tomography revealed space-occupying lesions in all dogs. Lesion detection by post mortem computed tomography was similar to lesion detection in autopsy for lung tissue (9 cases in computed tomography / 8 cases in autopsy), thoracic lymph nodes (9/8), spleen (6/7), kidney (2/2) and bone (3/3). Hepatic nodules, however, were difficult to detect with our scanning protocol (2/7). Histology of the core needle biopsies provided definitive diagnoses of histiocytic sarcoma in ten dogs, including confirmation by immunohistochemistry in six dogs. The biopsy samples of the remaining dog did not contain any identifiable neoplastic cells. Autolysis was the main reason for uncertain histological diagnoses. Conclusions: Post mortem computed tomography is a fast and effective method for the detection of lesions suspicious for histiocytic sarcoma in pulmonary, thoracic lymphatic, splenic, osseous and renal tissue. Optimization of the procedure regarding the scanning protocol and tissue sample size and number will improve the accuracy of the method. Keywords: Post mortem computed tomography, Core needle biopsy, Bernese mountain dog, Histiocytic sarcoma, Autopsy

Mothers adjust egg size to helper number in a cooperatively breeding cichlid

Mothers should adjust the size of propagules to the selective forces to which these offspring will be exposed. Usually, a larger propagule size is favored when young are exposed to high mortality risk or conspecific competition. Here we test 2 predictions on how egg size should vary with these selective agents. When offspring are cared for by parents and/or alloparents, protection may reduce the predation risk to young, which may allow mothers to invest less per single offspring. In the cooperatively breeding cichlid Neolamprologus pulcher, brood care helpers protect group offspring and reduce the latters' mortality rate. Therefore, females are expected to reduce their investment per egg when more helpers are present. In a first experiment, we tested this prediction by manipulating the helper number. In N. pulcher, helpers compete for dispersal opportunities with similar-sized individuals of neighboring groups. If the expected future competition pressure on young is high, females should increase their investment per offspring to give them a head start. In a second experiment, we tested whether females produce larger eggs when perceived neighbor density is high. Females indeed reduced egg size with increasing helper number. However, we did not detect an effect of local density on egg size, although females took longer to produce the next clutch when local density was high. We argue that females can use the energy saved by adjusting egg size to reduced predation risk to enhance future reproductive output. Adaptive adjustment of offspring size to helper number may be an important, as yet unrecognized, strategy of cooperative breeders.

Size parameters, size-class distribution and area-number relationship of microscopic charcoal: relevance for fire reconstruction

Charcoal analysis was conducted on sediment cores from three lakes to assess the relationship between the area and number of charcoal particles. Three charcoal-size parameters (maximum breadth, maximum length and area) were measured on sediment samples representing various vegetation types, including shrub tundra, boreal forest and temperate forest. These parameters and charcoal size-class distributions do not differ statistically between two sites where the same preparation technique (glycerine pollen slides) was used, but they differ for the same core when different techniques were applied. Results suggest that differences in charcoal size and size-class distribution are mainly caused by different preparation techniques and are not related to vegetation-type variation. At all three sites, the area and number concentrations of charcoal particles are highly correlated in standard pollen slides; 82–83% of the variability of the charcoal-area concentration can be explained by the particle-number concentration. Comparisons between predicted and measured area concentrations show that regression equations linking charcoal number and area concentrations can be used across sites as long as the same pollen-preparation technique is used. Thus it is concluded that it is unnecessary to measure charcoal areas in standard pollen slides – a time-consuming and tedious process.

Hepatic-portal vein infusions of glucagon-like peptide-1 reduce meal size and increase c-Fos expression in the nucleus tractus solitarii, area postrema and central nucleus of the amygdala in rats

We recently reported that brief, remotely controlled intrameal hepatic-portal vein infusions of glucagon-like peptide-1 (GLP-1) reduced spontaneous meal size in rats. To investigate the neurobehavioural correlates of this effect, we equipped male Sprague-Dawley rats with hepatic-portal vein catheters and assessed (i) the effect on eating of remotely triggered infusions of GLP-1 (1 nmol/kg, 5 min) or vehicle during the first nocturnal meal after 3 h of food deprivation and (ii) the effect of identical infusions performed at dark onset on c-Fos expression in several brain areas involved in the control of eating. GLP-1 reduced (P < 0.05) the size of the first nocturnal meal and increased its satiety ratio. Also, GLP-1 increased (P < 0.05) the number of c-Fos-expressing cells in the nucleus tractus solitarii, the area postrema and the central nucleus of the amygdala, but not in the arcuate or paraventricular hypothalamic nuclei. These data suggest that the nucleus tractus solitarii, the area postrema and the central nucleus of the amygdala play a role in the eating-inhibitory actions of GLP-1 infused into the hepatic-portal vein; it remains to be established whether activation of these brain nuclei reflect satiation, aversion, or both.

Heterogeneity in vaccination coverage explains the size and occurrence of measles epidemics in German surveillance data

The objective of this study was to characterize empirically the association between vaccination coverage and the size and occurrence of measles epidemics in Germany. In order to achieve this we analysed data routinely collected by the Robert Koch Institute, which comprise the weekly number of reported measles cases at all ages as well as estimates of vaccination coverage at the average age of entry into the school system. Coverage levels within each federal state of Germany are incorporated into a multivariate time-series model for infectious disease counts, which captures occasional outbreaks by means of an autoregressive component. The observed incidence pattern of measles for all ages is best described by using the log proportion of unvaccinated school starters in the autoregressive component of the model.

Quality, size, and composition of pediatric endobronchial biopsies in cystic fibrosis

BACKGROUND: Studies on airway remodeling in children with cystic fibrosis (CF) may be hampered by difficulty in obtaining evaluable endobronchial biopsy specimens because of large amounts of mucus and inflammation in the CF airway. We prospectively assessed how the quality of biopsy specimens obtained from children with CF compare with those from children with other airway diseases. METHODS: Fiberoptic bronchoscopy with endobronchial biopsy was performed in 67 CF children (age range, 0.2 to 16.8 years), 34 children with wheeze/asthma (W/A), and 64 control children with chronic respiratory symptoms. Up to three biopsy specimens were taken and stained with hematoxylin and eosin. Biopsy specimen size and structural composition were quantified using stereology. RESULTS: At least one evaluable biopsy specimen was obtained in 72% of CF children, in 79% of children with W/A, and in 72% of control subjects (difference was not significant). The use of large biopsy forceps (2.0 mm) rather than small biopsy forceps (1.0 mm) [odds ratio (OR), 5.8; 95% confidence interval (CI), 1.1 to 29.8; p = 0.037] and the number of biopsy specimens taken (odds ratio, 2.6; 95% confidence interval, 1.3 to 5.2; p = 0.006) significantly contributed to the success rate. Biopsy size and composition were similar between groups, except that CF children and those patients with W/A had a higher percentage of the biopsy specimen composed of muscle than did control subjects (median 6.2% and 9.7% vs 0.9%, respectively; p = 0.002). CONCLUSIONS: Biopsy size and quality are adequate for the study of airway remodeling in CF children as young as 2 months of age. Researchers should use large forceps when possible and take at least two biopsy specimens per patient. An increased airway smooth muscle content of the airway mucosa may contribute to the pathophysiology of CF lung disease.

Ring size of somatostatin analogues (ODT-8) modulates receptor selectivity and binding affinity

The synthesis, biological testing, and NMR studies of several analogues of H-c[Cys (3)-Phe (6)-Phe (7)-DTrp (8)-Lys (9)-Thr (10)-Phe (11)-Cys (14)]-OH (ODT-8, a pan-somatostatin analogue, 1) have been performed to assess the effect of changing the stereochemistry and the number of atoms in the disulfide bridge on binding affinity. Cysteine at positions 3 and/or 14 (somatostatin numbering) were/was substituted with d-cysteine, norcysteine, D-norcysteine, homocysteine, and/or D-homocysteine. The 3D structure analysis of selected partially selective, bioactive analogues (3, 18, 19, and 21) was carried out in dimethylsulfoxide. Interestingly and not unexpectedly, the 3D structures of these analogues comprised the pharmacophore for which the analogues had the highest binding affinities (i.e., sst 4 in all cases).

Prognostic factors in lymph node metastases of prostatic cancer patients: the size of the metastases but not extranodal extension independently predicts survival

AIMS: To analyse tumour characteristics and the prognostic significance of prostatic cancers with extranodal extension of lymph node metastases (ENE) in 102 node-positive, hormone treatment-naive patients undergoing radical prostatectomy and extended lymphadenectomy. METHODS AND RESULTS: The median number of nodes examined per patient was 21 (range 9-68), and the median follow-up time was 92 months (range 12-191). ENE was observed in 71 patients (70%). They had significantly more, larger and less differentiated nodal metastases, paralleled by significantly larger primary tumours at more advanced stages and with higher Gleason scores than patients without ENE. ENE defined a subgroup with significantly decreased biochemical recurrence-free (P = 0.038) and overall survival (P = 0.037). In multivariate analyses the diameter of the largest metastasis and Gleason score of the primary tumour were independent predictors of survival. CONCLUSIONS: ENE in prostatic cancer is an indicator lesion for advanced/aggressive tumours with poor outcome. However, the strong correlation with larger metastases suggests that ENE may result from their size, which was the only independent risk factor in the metastasizing component. Consequently, histopathological reports should specify the true indicator of poor survival in the lymphadenectomy specimens, which is the size of the largest metastasis in each patient.

The impact of different nanoparticle surface chemistry and size on uptake and toxicity in a murine macrophage cell line

This study investigated the uptake, kinetics and cellular distribution of different surface coated quantum dots (QDs) before relating this to their toxicity. J774.A1 cells were treated with organic, COOH and NH2 (PEG) surface coated QDs (40 nM). Model 20 nm and 200 nm COOH-modified coated polystyrene beads (PBs) were also examined (50 microg ml(-1)). The potential for uptake of QDs was examined by both fixed and live cell confocal microscopy as well as by flow cytometry over 2 h. Both the COOH 20 nm and 200 nm PBs were clearly and rapidly taken up by the J774.A1 cells, with uptake of 20 nm PBs being relatively quicker and more extensive. Similarly, COOH QDs were clearly taken up by the macrophages. Uptake of NH2 (PEG) QDs was not detectable by live cell imaging however, was observed following 3D reconstruction of fixed cells, as well as by flow cytometry. Cells treated with organic QDs, monitored by live cell imaging, showed only a small amount of uptake in a relatively small number of cells. This uptake was insufficient to be detected by flow cytometry. Imaging of fixed cells was not possible due to a loss in cell integrity related to cytotoxicity. A significant reduction (p<0.05) in the fluorescent intensity in a cell-free environment was found with organic QDs, NH2 (PEG) QDs, 20 nm and 200 nm PBs at pH 4.0 (indicative of an endosome) after 2 h, suggesting reduced stability. No evidence of exocytosis was found over 2 h. These findings confirm that surface coating has a significant influence on the mode of NP interaction with cells, as well as the subsequent consequences of that interaction.

Trial-to-trial size variability of motor-evoked potentials. A study using the triple stimulation technique

Motor-evoked potentials (MEPs) vary in size from one stimulus to the next. The objective of this study was to determine the cause and source of trial-to-trial MEP size variability. In two experiments involving 10 and 14 subjects, the variability of MEPs to cortical stimulation (cortical-MEPs) in abductor digiti minimi (ADM) and abductor hallucis (AH) was compared to those responses obtained using the triple stimulation technique (cortical-TST). The TST eliminates the effects of motor neuron (MN) response desynchronization and of repetitive MN discharges. Submaximal stimuli were used in both techniques. In six subjects, cortical-MEP variability was compared to that of brainstem-MEP and brainstem-TST. Variability was greater for MEPs than that for TST responses, by approximately one-third. The variability was the same for cortical- and brainstem-MEPs and was similar in ADM and AH. Variability concerned at least 10-15% of the MN pool innervating the target muscle. With the stimulation parameters used, repetitive MN discharges did not influence variability. For submaximal stimuli, approximately two-third of the observed MEP size variability is caused by the variable number of recruited alpha-MNs and approximately one-third by changing synchronization of MN discharges. The source of variability is most likely localized at the spinal segmental level.