Loss of the RhoGAP SRGP-1 promotes the clearance of dead and injured cells in Caenorhabditis elegans

Multicellular animals rapidly clear dying cells from their bodies. Many of the pathways that mediate this cell removal are conserved through evolution. Here, we identify srgp-1 as a negative regulator of cell clearance in both Caenorhabditis elegans and mammalian cells. Loss of srgp-1 function results in improved engulfment of apoptotic cells, whereas srgp-1 overexpression inhibits apoptotic cell corpse removal. We show that SRGP-1 functions in engulfing cells and functions as a GTPase activating protein (GAP) for CED-10 (Rac1). Interestingly, loss of srgp-1 function promotes not only the clearance of already dead cells, but also the removal of cells that have been brought to the verge of death through sublethal apoptotic, necrotic or cytotoxic insults. In contrast, impaired engulfment allows damaged cells to escape clearance, which results in increased long-term survival. We propose that C. elegans uses the engulfment machinery as part of a primitive, but evolutionarily conserved, survey mechanism that identifies and removes unfit cells within a tissue.     

Isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 μm in diameter can be observed and are ready for further passaging and expansion.     

Comparison of ISSR polymorphism among cattle breeds

Polymorphism analysis of DNA fragments flanked by (AG)₉C and (GA)₉C inverted dinucleotide microsatellite repeats in 766 animals of 19 cattle breeds and one breeding type revealed 66 fragments, of which 64 were polymorphic. The breeds proved to differ in the frequency and presence or absence of amplified DNA fragments at the genomic level, indicating that ISSR fingerprinting is informative for differentiating the PCR product spectra and cattle breeds. Multilocus ISSR polymorphism analysis identified the group of fragments that can be used as Bos taurus and B. indicus species markers to describe the standards of breeds, their genetic profiles, and breed-specific patterns. Based on ISSR polymorphism, a prototypal gene pool of cattle was constructed and the breeds closest to it were identified. Genetic diversity analysis made it possible to assume that an optimal mean heterozygosity is characteristic of cattle breeds and that deviations from this optimum are indicative of various processes occurring in the population (breed).     

Effects of different photoperiods and handling stress on spawning and reproductive performance of pikeperch Sander lucioperca

The objective of this study was to control the reproductive cycle of pikeperch (Sander lucioperca) through determining the effects of different photoperiods and handling stress on the reproduction quality, timing and quality of spawning, fertilization, sex steroids, and cortisol concentrations. In this study, 72 pikeperch broodstocks with an average weight of 1367 ± 55.3 g were exposed to different photoperiods including constant light (24L:0D), constant darkness (0L:24D), and 12 h of light, 12 h of darkness (12L:12D) for 40 days. Half of the broodstocks of each photoperiod treatment were exposed to handling stress at a specific time of the day. Applying different photoperiods caused changes in the timing of broodstocks' spawning, so that fish under 24L:0D spawned earlier than those of other photoperiods, and stressed fish of the 0L:24D photoperiod had a delayed spawning compared to others. Also, the spawning of the broodstocks at different photoperiods which were exposed to handling stress was either delayed or did not occur at all. The highest and lowest spawnings were observed in the morning and at night, respectively. Fertilization percentage, number of eggs per gram, sex steroids including estradiol, progesterone, and testosterone, as well as cortisol and calcium concentrations did not show any significant difference in different photoperiods and handling stress. In stressed males of the 24L:0D photoperiod, there only was a significant decrease of testosterone concentration compared to the beginning of the experiment. Results indicated that the spawning performance of pikeperch broodstocks could be considerably stimulated using an effective photoperiod. Similarly, pikeperch broodstocks in culture systems are usually affected by handling stress, and this stress could lead to a poor reproductive performance and inhibition of spawning.     

Correlation of the amino-acid sequence and the 3D structure of the functional domain of EmaA from Aggregatibacter actinomycetemcomitans

Adhesion to collagen is an important virulence determinant for the periodontal pathogen Aggregatibacter actinomycetemcomitans. Binding to collagen is mediated by the extracellular-matrix protein adhesin-A (EmaA). EmaA is a homotrimeric autotransporter protein that forms flexible antenna-like appendages on the bacterium surface. An ellipsoidal structure at the distal end of the appendage, composed of three subdomains, contains the functional domain of the molecule. A correlation between amino-acid sequence and subdomain structure (SI and SII) was proposed based on an analysis of the volume/molecular weight ratio. EmaA from three mutant strains (deletions of amino-acids 70-206 and 70-386 and a substitution mutation G162S) has been studied by electron microscopy to test this hypothesis. 3D structures were analyzed using single-axis tilt tomography of negatively stained preparations of bacteria combined with subvolume averaging. Additionally, a large number of 2D images of the apical domain of the adhesins from the mutants were extracted from micrographs of the bacterial surface, aligned and classified. The combined data showed that amino-acids 70-206 localize to subdomain SI and 70-386 comprise subdomains SI and SII. Moreover, we showed that the substitution mutation G162S, which abolishes collagen binding activity, does not affect the overall structural integrity of the functional domain. However, the structure of subdomain SI in this mutant is slightly altered with respect to the wild-type strain. These data also have allowed us to interpret the architectural features of each subdomain of EmaA in more detail and to correlate the 3D structure of the functional domain of EmaA with the amino-acid sequence.     

Benchmarking academic plastic surgery services in the United States

Rising health care costs and increasingly demanding patients are only some of the challenges faced by academic plastic surgery services in their pursuit of excellence in education, research, and patient care. Benchmarking, when correctly applied, is a powerful tool that can help services learn from each other's experiences. This study aimed at creating the first benchmarking report summarizing performance indicators and management practices of some of the most complete academic plastic surgery units in the United States. Results provide an opportunity for plastic surgery leaders to benchmark against their own units, identify eventual gaps, and improve their performance as needed.     

Phagocytic signaling: you can touch, but you can't eat

The ability of phagocytes to discriminate between viable/healthy and apoptotic/foreign/abnormal cells is of fundamental importance; a recent study provides new molecular insights into the function of CD47-SIRP alpha signaling in this discrimination.     

Reversible denaturation of carbonic anhydrase provides a method for its adsorptive immobilization

Palmityl-substituted sepharose 4B has been used for adsorptive immobilization of heat-denatured carbonic anhydrase. The native form of this enzyme does not show any affinity for binding to this hydrophobic support. However, through the process of denaturation-renaturation performed by heating and subsequent cooling of an enzyme solution in the presence of the matrix, it was possible to obtain a catalytically active immobilized preparation, which was used successfully in continuous catalytic transformations. It is suggested that this simple procedure may provide a convenient method of immobilization for proteins, which are not normally adsorbed on hydrophobic supports.     

Allelic polymorphism of kappa-casein gene (CSN3) in Russian cattle breeds and its informative value as a genetic marker

The frequencies of the kappa-casein gene (CSN3) alleles and genotypes have been determined in five Russian cattle breeds (Bestuzhev, Kalmyk, Russian Black Pied, Yaroslavl, and Yakut breeds) by means of PCR-RFLP analysis using two independent restriction nucleases (HinfI and TaqI) and by allele-specific PCR. Typing alleles A and B of CSN3 is of practical importance, because allele B is correlated with commercially valuable parameters of milk productivity (protein content and milk yield) and improves the cheese yielding capacity. The frequencies of the B allele of CSN3 in the breeds studied vary from 0.16 to 0.50; and those of the AB and BB genotypes, from 0.27 to 0.60 and from 0.02 to 0.23, respectively. The Yaroslavl breed had the highest frequencies of CSN3 allele B and genotype BB (0.50 and 0.23, respectively). The frequencies of the B allele and BB genotype in other breeds studied varied from 0.25 to 0.32 and from 0.03 to 0.09, respectively. In none of the breeds studied have the observed and expected heterozygosities been found to differ from each other significantly. However, the observed genotype distributions significantly differ from the expected one in some herds (in most such cases, an excess of heterozygotes is observed). Two herds of the Yaroslavl breed dramatically differ from each other in the heterozygosity level: a deficit (D = -0.14) and an excess (D = 0.20) of heterozygotes have been observed at the Mikhailovskoe and Gorshikha farms, respectively. In general, however, the heterozygosity of the Yaroslavl breed corresponds to the expected level (D = 0.04). Analysis of breeds for homogeneity with the use of Kulback's test has shown that all cattle breeds studied are heterogeneous, the CSN3 diversity within breeds being higher than that among different breeds, which is confirmed by low Fst values (0.0025-0.0431). Thus, a DNA marker based on CSN3 gene polymorphism is extremely important for breeding practice as a marker of milk quality; however, it is inapplicable to marking differences between breeds or phylogenetic relationships between cattle breeds because of the high diversity with respect to this locus within breeds.     

Dock180 and ELMO1 proteins cooperate to promote evolutionarily conserved Rac-dependent cell migration

Cell migration is essential throughout embryonic and adult life. In numerous cell systems, the small GTPase Rac is required for lamellipodia formation at the leading edge and movement ability. However, the molecular mechanisms leading to Rac activation during migration are still unclear. Recently, a mammalian superfamily of proteins related to the prototype member Dock180 has been identified with homologues in Drosophila and Caenorhabditis elegans. Here, we addressed the role of Dock180 and ELMO1 proteins, which function as a complex to mediate Rac activation, in mammalian cell migration. Using mutants of Dock180 and ELMO1 in a Transwell assay as well as transgenic rescue of a C. elegans mutant lacking CED-5 (Dock180 homologue), we identified specific regions of Dock180 and ELMO1 required for migration in vitro and in a whole animal model. In both systems, the Dock180.ELMO1 complex formation and the ability to activate Rac were required. We also found that ELMO1 regulated multiple Dock180 superfamily members to promote migration. Interestingly, deletion mutants of ELMO1 missing their first 531 or first 330 amino acids that can still bind and cooperate with Dock180 in Rac activation failed to promote migration, which correlated with the inability to localize to lamellipodia. This finding suggests that Rac activation by the ELMO.Dock180 complex at discrete intracellular locations mediated by the N-terminal 330 amino acids of ELMO1 rather than generalized Rac activation plays a role in cell migration.