The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Rates of readmission and death associated with leaving hospital against medical advice: a population-based study

Background:

Leaving hospital against medical advice may have adverse consequences. Previous studies have been limited by evaluating specific types of patients, small sample sizes and incomplete determination of outcomes. We hypothesized that leaving hospital against medical advice would be associated with increases in subsequent readmission and death.

Methods:

In a population-based analysis involving all adults admitted to hospital and discharged alive in Manitoba from Apr. 1, 1990, to Feb. 28, 2009, we evaluated all-cause 90-day mortality and 30-day hospital readmission. We used multivariable regression, adjusted for age, sex, socioeconomic status, year of hospital admission, patient comorbidities, hospital diagnosis, past frequency of admission to hospital, having previously left hospital against medical advice and data clustering (patients with multiple admissions). For readmission, we assessed both between-person and within-person effects of leaving hospital against medical advice.

Results:

Leaving against medical advice occurred in 21 417 of 1 916 104 index hospital admissions (1.1%), and was associated with higher adjusted rates of 90-day mortality (odds ratio [OR] 2.51, 95% confidence interval [CI] 2.18–2.89), and 30-day hospital readmission (within-person OR 2.10, CI 1.99–2.21; between-person OR 3.04, CI 2.79–3.30). In our additional analyses, elevated rates of readmission and death associated with leaving against medical advice were manifest within 1 week and persisted for at least 180 days after discharge.

Interpretation:

Adults who left the hospital against medical advice had higher rates of hospital readmission and death. The persistence of these effects suggests that they are not solely a result of incomplete treatment of acute illness. Interventions aimed at reducing these effects may need to include longitudinal interventions extending beyond admission to hospital.Patients leaving hospital against medical advice have been discussed in the medical literature for more than 50 years.¹ Reported to occur in 1%–2% of patients in general hospitals,^2,3 the numbers are large; in the United States, 368 000 patients left against medical advice in 2007,³ and rates higher than 10% have been documented in certain subgroups, including Canadian patients with HIV and predominantly poor residents of inner city areas.^4,5 The main concern over leaving hospital against medical advice is that it may increase morbidity or mortality. Previous studies attempting to assess this effect^2,4–13 have all been restricted to specific types of patients, and most studies were limited by small sample sizes and incomplete determination of outcomes. In this study, we used data that avoided these limitations to test the hypothesis that patients who leave hospital against medical advice have higher rates of hospital readmission and death.     

How many physicians does Canada need to care for our aging population?

BACKGROUND: There is concern that the aging of Canada''s population will strain our health care system. The authors address this concern by examining changes in the physician supply between 1986 and 1994 and by assessing the availability of physicians in 1994 relative to population growth and aging, and relative to supply levels in the benchmark province of Alberta. METHODS: Physician numbers were obtained from the Canadian Institute for Health Information. The amount of services provided by each specialty to each patient age group was analysed using Manitoba physician claims data. Population growth statistics were obtained from Statistics Canada. Age- and specialty-specific utilization data and age-specific population growth patterns were used to estimate the number and type of physicians that would have been required in each province to keep up with population growth between 1986 and 1994, in comparison with actual changes in the physician numbers. Physician supply in Alberta was used as a benchmark against which other provinces were measured. RESULTS: Overall, Canada''s physician supply between 1986 and 1994 kept pace with population growth and aging. Some specialties grew much faster than population changes warranted, whereas others grew more slowly. By province, the supply of general practitioners (GPs) grew much faster than the population served in New Brunswick (16.6%), Alberta (6.5%) and Quebec (5.3%); the GP supply lagged behind in Prince Edward Island (-5.4%). Specialist supply outpaced population growth substantially in Nova Scotia (10.4%), Newfoundland (8.5%), New Brunswick (7.3%) and Saskatchewan (6.8%); it lagged behind in British Columbia (-9.2%). Using Alberta as the benchmark resulted in a different assessment: Newfoundland (15.5%) and BC (11.7%) had large surpluses of GPs by 1994, whereas PEI (-21.1%), New Brunswick (-14.8%) and Manitoba (-11.1%) had substantial deficits; Quebec (37.3%), Ontario (24.0%), Nova Scotia (11.6%), Manitoba (8.2%) and BC (7.6%) had large surpluses of specialists by 1994, whereas PEI (-28.6%), New Brunswick (-25.9%) and Newfoundland (-23.8%) had large deficits. INTERPRETATION: The aging of Canada''s population poses no threat of shortage to the Canadian physician supply in general, nor to most specialist groups. The marked deviations in provincial physician supply from that of the benchmark province challenge us to understand the costs and benefits of variations in physician resources across Canada and to achieve a more equitable needs-based availability of physicians within provinces and across the country.     

Porcine reproductive and respiratory syndrome virus (PRRSV) infection spreads by cell-to-cell transfer in cultured MARC-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection

Background

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145) cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA) analyses of PRRSV infection of cultured MARC-145 cells were carried out in experiments designed to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs.

Results

Flow cytometric and FA analyses of PRRSV antigen expression revealed distinct primary and secondary phases of MARC-145 cell infection. PRRSV antigen was randomly expressed in a few percent of cells during the primary phase of infection (up to about 20–22 h p.i.), but the logarithmic infection phase (days 2–3 p.i.), was characterized by secondary spread to clusters of infected cells. The formation of secondary clusters of PRRSV-infected cells preceded the development of CPE in MARC-145 cells, and both primary and secondary PRRSV infection were inhibited by colchicine and cytochalasin D, demonstrating a critical role of the cytoskeleton in viral permissiveness as well as cell-to-cell transmission from a subpopulation of cells permissive for free virus to secondary targets. Cellular expression of actin also appeared to correlate with PRRSV resistance, suggesting a second role of the actin cytoskeleton as a potential barrier to cell-to-cell transmission. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-γ (IFN-γ), as well as the more-potent experimental antiviral agent AK-2.

Conclusion

The results demonstrate two distinct mechanisms of PRRSV infection: primary infection of a relatively small subpopulation of innately PRRSV-permissive cells, and secondary cell-to-cell transmission to contiguous cells which appear non-permissive to free virus. The results also indicate that an intact cytoskeleton is critical for PRRSV infection, and that viral permissiveness is a highly efficient drug target to control PRRSV infection. The data from this experimental system have important implications for the mechanisms of PRRSV persistence and pathology, as well as for a better understanding of arterivirus regulation.     

Antibacterial activity against β- lactamase producing Methicillin and Ampicillin-resistants Staphylococcus aureus: fractional Inhibitory Concentration Index (FICI) determination

Background

The present study reports the antibacterial capacity of alkaloid compounds in combination with Methicillin and Ampicillin-resistants bacteria isolated from clinical samples. The resistance of different bacteria strains to the current antibacterial agents, their toxicity and the cost of the treatment have led to the development of natural products against the bacteria resistant infections when applied in combination with conventional antimicrobial drugs.

Method

The antibacterial assays in this study were performed by using inhibition zone diameters, MIC, MBC methods, the time-kill assay and the Fractional Inhibitory Concentration Index (FICI) determination. On the whole, fifteen Gram-positive bacterial strains (MRSA/ARSA) were used. Negative control was prepared using discs impregnated with 10 % DMSO in water and commercially available Methicillin and Ampicillin from Alkom Laboratories LTD were used as positive reference standards for all bacterial strains.

Results

We noticed that the highest activities were founded with the combination of alkaloid compounds and conventional antibiotics against all bacteria strains. Then, results showed that after 7 h exposition there was no viable microorganism in the initial inoculums.

Conclusion

The results of this study showed that alkaloid compounds in combination with conventional antibiotics (Methicillin, Ampicillin) exhibited antimicrobial effects against microorganisms tested. These results validate the ethno-botanical use of Cienfuegosia digitata Cav. (Malvaceae) in Burkina Faso. Moreover, this study demonstrates the potential of this herbaceous as a source of antibacterial agent that could be effectively used for future health care purposes.     

Supply management of high-value components with a credit constraint

Due to rapid increases in the automotive markets in emerging economies, leading car manufacturers rapidly expand their manufacturing capacity in countries such as China. A certain portion of key performance components however continue to be sourced from developed countries. We study such a manufacturer that imports high-value components from a developed country. There are two available transportation modes: a slow mode with low cost and long and stochastic lead time, and a fast mode with high cost and short and deterministic lead time. Moreover, the manufacturer is subject to a credit constraint that bounds both the in-warehouse inventory and the number of outstanding orders (because it needs to pay for the components in advance). Consequently, the cheapest hedge against demand and supply uncertainty??inventory??is only available to a limited extent and the decision maker must turn to using the fast transportation mode for a much larger share of the orders. We model the manufacturer??s ordering policy and study its performance using simulation. Our study shows the adverse effects that the credit limit has on the growth opportunities of such companies in developing countries that import high-value components or other goods from developed countries. We show that especially the reduction in lead time variability can substantially reduce these adverse effects. Realizing that this variability is primarily caused by the import customs procedures, governments in developing economies have a means to assist their local manufacturers.     

Genome-wide association and genomic prediction for host response to porcine reproductive and respiratory syndrome virus infection

Background

Host genetics has been shown to play a role in porcine reproductive and respiratory syndrome (PRRS), which is the most economically important disease in the swine industry. A region on Sus scrofa chromosome (SSC) 4 has been previously reported to have a strong association with serum viremia and weight gain in pigs experimentally infected with the PRRS virus (PRRSV). The objective here was to identify haplotypes associated with the favorable phenotype, investigate additional genomic regions associated with host response to PRRSV, and to determine the predictive ability of genomic estimated breeding values (GEBV) based on the SSC4 region and based on the rest of the genome. Phenotypic data and 60 K SNP genotypes from eight trials of ~200 pigs from different commercial crosses were used to address these objectives.

Results

Across the eight trials, heritability estimates were 0.44 and 0.29 for viral load (VL, area under the curve of log-transformed serum viremia from 0 to 21 days post infection) and weight gain to 42 days post infection (WG), respectively. Genomic regions associated with VL were identified on chromosomes 4, X, and 1. Genomic regions associated with WG were identified on chromosomes 4, 5, and 7. Apart from the SSC4 region, the regions associated with these two traits each explained less than 3% of the genetic variance. Due to the strong linkage disequilibrium in the SSC4 region, only 19 unique haplotypes were identified across all populations, of which four were associated with the favorable phenotype. Through cross-validation, accuracies of EBV based on the SSC4 region were high (0.55), while the rest of the genome had little predictive ability across populations (0.09).

Conclusions

Traits associated with response to PRRSV infection in growing pigs are largely controlled by genomic regions with relatively small effects, with the exception of SSC4. Accuracies of EBV based on the SSC4 region were high compared to the rest of the genome. These results show that selection for the SSC4 region could potentially reduce the effects of PRRS in growing pigs, ultimately reducing the economic impact of this disease.     

Slow response of soil organic matter to the reduction in atmospheric nitrogen deposition in a Norway spruce forest

Global nitrogen (N) deposition rates in terrestrial environments have quadrupled since preindustrial times, causing structural and functional changes of ecosystems. Different emission reduction policies were therefore devised. The aim of our study was to investigate if, and over what timescale, processes of soil organic matter (OM) transformation respond to a decline in atmospheric N deposition. A N‐saturated spruce forest (current N deposition: 34 kg ha^?1 yr^?1; critical N load: 14 kg ha^?1 yr^?1), where N deposition has been reduced to 11.5 kg ha^?1 yr^?1 since 1991, was studied. Besides organic C and organic and inorganic N, noncellulosic carbohydrates, amino sugars and amino acids were determined. A decline in organic N in litter indicated initial effects at plant level. However, there were no changes in biomarkers upon the reduction in N deposition. In addition, inorganic N was not affected by reduced N deposition. The results showed that OM cycling and transformation processes have not responded so far. It was concluded that no direct N deposition effects have occurred due to the large amount of stored organic N, which seems to compensate for the reduction in deposited N. Obviously, the time span of atmospheric N reduction (about 14.5 years) is too short compared with the mean turnover time of litter to cause indirect effects on the composition of organic C and N compounds. It is assumed that ecological processes, such as microbial decomposition or recycling of organic N and C, react slowly, but may start within the next decade with the incorporation of the new litter.