Plaque2.0—A High-Throughput Analysis Framework to Score Virus-Cell Transmission and Clonal Cell Expansion

Classical plaque assay measures the propagation of infectious agents across a monolayer of cells. It is dependent on cell lysis, and limited by user-specific settings and low throughput. Here, we developed Plaque2.0, a broadly applicable, fluorescence microscopy-based high-throughput method to mine patho-biological clonal cell features. Plaque2.0 is an open source framework to extract information from chemically fixed cells by immuno-histochemistry or RNA in situ hybridization, or from live cells expressing GFP transgene. Multi-parametric measurements include infection density, intensity, area, shape or location information at single plaque or population levels. Plaque2.0 distinguishes lytic and non-lytic spread of a variety of DNA and RNA viruses, including vaccinia virus, adenovirus and rhinovirus, and can be used to visualize simultaneous plaque formation from co-infecting viruses. Plaque2.0 also analyzes clonal growth of cancer cells, which is relevant for cell migration and metastatic invasion studies. Plaque2.0 is suitable to quantitatively analyze virus infections, vector properties, or cancer cell phenotypes.     

Ceftiofur derivates in serum and endometrial tissue after intramuscular administration in healthy mares

Endometritis is one of the major problems in the horse breeding industry. The use of antibiotics for treatment of endometritis in the mare is recommended as best practice. The intrauterine application of antibiotics, however, has been under discussion over the last years because of concerns about its efficacy. The systemic use of antibiotics has been considered more effective because of its better distribution within the uterus. The objective of the present study was to determine the concentration of ceftiofur derivates in serum and endometrial tissue after intramuscular administration. Specifically, the authors tested the hypothesis that ceftiofur concentrations in serum and endometrial tissue remain above the minimum inhibitory concentration (MIC) for common uterine pathogens for 24 h. Nine mares in estrus received a single dose of 2.2 mg/kg ceftiofur hydrochloride intramuscular per kg of body weight. Blood samples and endometrial tissue were obtained immediately before treatment (−1 h) and 2 h and 24 h after treatment. Endometrial tissue was collected with a Kevorkian biopsy punch. Additional blood samples were collected 4 h and 10 h after treatment from the jugular veins. For determination of ceftiofur derivates in serum and endometrial tissue a high performance liquid chromatography (HPLC) assay was used. Results in serum and uterine tissue revealed greatest concentration of ceftiofur at 2 h and lowest concentrations at 24 h after treatment. Concentrations of ceftiofur at 2 and 24 h after treatment were significantly greater in serum than in endometrial tissue, but remained above the reported MIC for Streptococcus equi zooepidemicus and Escherichia coli in both serum and endometrial tissue until 24 h after treatment.     

Replication of recently identified systemic lupus erythematosus genetic associations: a case–control study

Introduction

We aimed to replicate association of newly identified systemic lupus erythematosus (SLE) loci.

Methods

We selected the most associated SNP in 10 SLE loci. These 10 SNPs were analysed in 1,579 patients with SLE and 1,726 controls of European origin by single-base extension. Comparison of allele frequencies between cases and controls was done with the Mantel–Haenszel approach to account for heterogeneity between sample collections.

Results

A previously controversial association with a SNP in the TYK2 gene was replicated (odds ratio (OR) = 0.79, P = 2.5 × 10-⁵), as well as association with the X chromosome MECP2 gene (OR = 1.26, P = 0.00085 in women), which had only been reported in a single study, and association with four other loci, 1q25.1 (OR = 0.81, P = 0.0001), PXK (OR = 1.19, P = 0.0038), BANK1 (OR = 0.83, P = 0.006) and KIAA1542 (OR = 0.84, P = 0.001), which have been identified in a genome-wide association study, but not found in any other study. All these replications showed the same disease-associated allele as originally reported. No association was found with the LY9 SNP, which had been reported in a single study.

Conclusions

Our results confirm nine SLE loci. For six of them, TYK2, MECP2, 1q25.1, PXK, BANK1 and KIAA1542, this replication is important. The other three loci, ITGAM, STAT4 and C8orf13-BLK, were already clearly confirmed. Our results also suggest that MECP2 association has no influence in the sex bias of SLE, contrary to what has been proposed. In addition, none of the other associations seems important in this respect.     

BCR sequences essential for transformation by the BCR-ABL oncogene bind to the ABL SH2 regulatory domain in a non-phosphotyrosine-dependent manner.

BCR-ABL is a chimeric oncogene implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. BCR first exon sequences specifically activate the tyrosine kinase and transforming potential of BCR-ABL. We have tested the hypothesis that activation of BCR-ABL may involve direct interaction between BCR sequences and the tyrosine kinase regulatory domains of ABL. Full-length c-BCR as well as BCR sequences retained in BCR-ABL bind specifically to the SH2 domain of ABL. The binding domain has been localized within the first exon of BCR and consists of at least two SH2-binding sites. This domain is essential for BCR-ABL-mediated transformation. Phosphoserine/phosphothreonine but not phosphotyrosine residues on BCR are required for interaction with the ABL SH2 domain. These findings extend the range of potential SH2-protein interactions in growth control pathways and suggest a function for SH2 domains in the activation of the BCR-ABL oncogene as well as a role for BCR in cellular signaling pathways.     

Raiding and emigration dynamics in the ponerine army ant Leptogenys distinguenda (Hymenoptera, Formicidae)

Summary: Field and laboratory observations demonstrate that Leptogenys distinguenda is characterized by typical army ant behavior. Like in the "classical" army ants from the subfamilies Ecitoninae, Dorylinae and Aenictinae, raiding and emigration behavior are closely linked. The direction of raids can be altered in field experiments to a wide extent by offering ample food, suggesting it is highly influenced by the patchiness of prey. The sum of recruitments coming from one direction and the recruitment overrun are primarily responsible for the spatial development and the extension of raids. Emigration frequency can be suppressed by overfeeding a colony in the field. This result is interpreted as a secondary effect of reduced swarming activity, which gets suppressed as well in the same experiment. The discovery of a suitable nest site is considered the proximate stimulus for emigration, and the best explored areas are those that have been most thoroughly raided. As a result, emigrations are likely to lead into areas with high prey densities.     

Exercise decreases speedball self-administration

Aims

Epidemiological studies report that individuals who exercise are less likely to abuse drugs. Preclinical studies report that exercise, in the form of treadmill or wheel running, reliably decreases the self-administration of psychomotor stimulants and opioids. To date, preclinical studies have only examined the effects of exercise on responding maintained by individual drugs and not by combinations of multiple drugs. This limits the translational appeal of these studies because polydrug abuse is common among substance abusing populations. The purpose of this study was to examine the effects of exercise on the self-administration of speedball, a combination of cocaine and heroin that is frequently encountered in intravenous drug abusing populations.

Main methods

Female rats were obtained at weaning and assigned to sedentary or exercising conditions. Sedentary rats were housed in standard cages that permitted no exercise beyond normal cage ambulation; exercising rats were housed in similar cages with an activity wheel. After 6 weeks, rats were implanted with intravenous catheters and trained to self-administer cocaine, heroin, and dose combinations of cocaine and heroin (i.e., speedball) on a progressive ratio schedule of reinforcement.

Key findings

Doses of speedball maintained greater levels of responding than corresponding doses of cocaine and heroin alone. Importantly, responding maintained by cocaine, heroin, and speedball was lower in exercising rats than sedentary rats.

Significance

These data indicate that exercise decreases the self-administration of speedball and suggest that exercise may reduce the abuse of drug combinations that have traditionally been resistant to treatment.     

Developmental expression of Magmas in murine tissues and its co-expression with the GM-CSF receptor.

Magmas is a protein that is involved in GM-CSF signaling in a myeloid cell line. Its precise role in the signal transduction process is unclear. To accurately characterize Magmas expression in a variety of cells, mouse embryos and adult murine tissues were analyzed for both mRNA and protein content. Magmas expression was detected as early as the day 6.5 embryo. The level of expression was developmentally regulated. During embryogenesis, elevated Magmas was observed in several structures, including heart, liver, notochord, choroid plexus, cervical ganglion, and nasal mucosa. Muscle, pancreas, intestinal mucosa, and testes were among the adult tissues with high Magmas expression. Most cell types, including hepatocytes and skeletal, smooth, and cardiac myocytes, also expressed the GM-CSF receptor (GMR) but the relative tissue levels of GMR were not always proportional to Magmas. The expression patterns suggest that Magmas has a role in both developing and mature tissues.     

Neuroprotection against CCl4 induced brain damage with crocin in Wistar rats

Owing to its lipophilic property, carbon tetrachloride (CCl₄) is rapidly absorbed by both the liver and brain. We investigated the protective effects of crocin against brain damage caused by CCl₄. Fifty rats were divided into five groups of ten: control, corn oil, crocin, CCl₄ and CCl₄ + crocin. CCl₄ administration decreased glutathione (GSH) and total antioxidant status (TAS) levels, and catalase (CAT) activity, while significant increases were observed in malondialdehyde (MDA) and total oxidant status (TOS) levels and superoxide dismutase (SOD) activity. The cerebral cortex nuclear lamina developed a spongy appearance, neuronal degeneration was observed in the hippocampus, and heterochromatic and pyknotic neurons with increased cytoplasmic eosinophilia were observed in the hippocampus after CCl₄ treatment. Because crocin exhibits strong antioxidant properties, crocin treatment increased GSH and TAS levels and CAT activities, and decreased MDA and TOS levels and SOD activity; significant improvements also were observed in histologic architecture. We found that crocin administration nearly eliminated CCl₄ induced brain damage by preventing oxidative stress.     

Characterization of B lymphocyte lineage progenitor cells from mice with severe combined immune deficiency disease (SCID) made possible by long term culture

A single gene mutation results in near absence of B and T lymphocytes and their immediate progenitors in mice with severe combined immunodeficiency disease (SCID). However, long term culture conditions allowed rapid outgrowth of lymphocytes from SCID bone marrow suspensions, and this permitted their detailed analysis. The cells were judged to be committed to the B lymphocyte lineage on the basis of expression of the BP-1 antigen, as well as by the density and pattern of expression of other markers. Cultured SCID lymphocytes were indistinguishable from control BALB/c cells in terms of morphology, typing for 13 cell surface markers, and changes in cell surface antigen expression with time in culture. In contrast to cultures of normal cells, which always included IgM synthesizing cells, SCID lymphocytes rarely expressed mu heavy chains. Southern blot analysis demonstrated that at least the first Ig gene rearrangement step had occurred in most of the cultured cells. The patterns of JH gene rearrangements suggested that relatively limited population diversity existed in individual cultures of SCID and normal BALB/c marrow. In addition, there was evidence that abnormal Ig heavy chain gene rearrangements had taken place in lymphocytes from approximately 25% of the SCID cultures. These cells were distinguished by the absence of detectable JH gene segments. kappa light chain genes appeared to be unrearranged in SCID cultured lymphocytes. We conclude that the lymphopoietic microenvironments of SCID mice are probably normal, and the animals have infrequent progenitors of B cells. Aberrant or nonproductive IgH gene rearrangements may account for the absence of pre-B and B cells in SCID mice. This study demonstrates the usefulness of long term culture methodology for isolating rare subsets of non-transformed lymphoid cells from normal and genetically defective hemopoietic tissues.