Use of chromomycin A3 staining in bovine sperm cells for detection of protamine deficiency

Sperm chromatin integrity is essential for accurate transmission of male genetic information, and normal sperm chromatin structure is important for fertilization. Protamine is a nuclear protein that plays a key role in sperm DNA integrity, because it is responsible for sperm DNA stability and packing until the paternal genome is delivered into the oocyte during fertilization. Our aim was to investigate protamine deficiency in sperm cells of Bos indicus bulls (Nelore) using chromomycin A₃ (CMA₃) staining. Frozen semen from 14 bulls were thawed, then fixed in Carnoy's solution. Smears were prepared and analyzed by microscopy. As a positive control of CMA₃ staining, sperm from one bull was subjected to deprotamination of nuclei. The percentage of CMA₃-positive bovine sperm did not vary among batches. Only two bulls showed a higher percentage of CMA₃-positive sperm cells compared to the others. CMA₃ is a simple and useful tool for detecting sperm protamine deficiency in bulls.     

Mapping the Shapes of Phylogenetic Trees from Human and Zoonotic RNA Viruses

A phylogeny is a tree-based model of common ancestry that is an indispensable tool for studying biological variation. Phylogenies play a special role in the study of rapidly evolving populations such as viruses, where the proliferation of lineages is constantly being shaped by the mode of virus transmission, by adaptation to immune systems, and by patterns of human migration and contact. These processes may leave an imprint on the shapes of virus phylogenies that can be extracted for comparative study; however, tree shapes are intrinsically difficult to quantify. Here we present a comprehensive study of phylogenies reconstructed from 38 different RNA viruses from 12 taxonomic families that are associated with human pathologies. To accomplish this, we have developed a new procedure for studying phylogenetic tree shapes based on the ‘kernel trick’, a technique that maps complex objects into a statistically convenient space. We show that our kernel method outperforms nine different tree balance statistics at correctly classifying phylogenies that were simulated under different evolutionary scenarios. Using the kernel method, we observe patterns in the distribution of RNA virus phylogenies in this space that reflect modes of transmission and pathogenesis. For example, viruses that can establish persistent chronic infections (such as HIV and hepatitis C virus) form a distinct cluster. Although the visibly ‘star-like’ shape characteristic of trees from these viruses has been well-documented, we show that established methods for quantifying tree shape fail to distinguish these trees from those of other viruses. The kernel approach presented here potentially represents an important new tool for characterizing the evolution and epidemiology of RNA viruses.     

A likelihood‐based approach for assessment of extra‐pair paternity and conspecific brood parasitism in natural populations

Genotypes are frequently used to assess alternative reproductive strategies such as extra‐pair paternity and conspecific brood parasitism in wild populations. However, such analyses are vulnerable to genotyping error or molecular artefacts that can bias results. For example, when using multilocus microsatellite data, a mismatch at a single locus, suggesting the offspring was not directly related to its putative parents, can occur quite commonly even when the offspring is truly related. Some recent studies have advocated an ad‐hoc rule that offspring must differ at more than one locus in order to conclude that they are not directly related. While this reduces the frequency with which true offspring are identified as not directly related young, it also introduces bias in the opposite direction, wherein not directly related young are categorized as true offspring. More importantly, it ignores the additional information on allele frequencies which would reduce overall bias. In this study, we present a novel technique for assessing extra‐pair paternity and conspecific brood parasitism using a likelihood‐based approach in a new version of program cervus . We test the suitability of the technique by applying it to a simulated data set and then present an example to demonstrate its influence on the estimation of alternative reproductive strategies.     

Basic and applied uses of genome‐scale metabolic network reconstructions of Escherichia coli

The genome‐scale model (GEM) of metabolism in the bacterium Escherichia coli K‐12 has been in development for over a decade and is now in wide use. GEM‐enabled studies of E. coli have been primarily focused on six applications: (1) metabolic engineering, (2) model‐driven discovery, (3) prediction of cellular phenotypes, (4) analysis of biological network properties, (5) studies of evolutionary processes, and (6) models of interspecies interactions. In this review, we provide an overview of these applications along with a critical assessment of their successes and limitations, and a perspective on likely future developments in the field. Taken together, the studies performed over the past decade have established a genome‐scale mechanistic understanding of genotype–phenotype relationships in E. coli metabolism that forms the basis for similar efforts for other microbial species. Future challenges include the expansion of GEMs by integrating additional cellular processes beyond metabolism, the identification of key constraints based on emerging data types, and the development of computational methods able to handle such large‐scale network models with sufficient accuracy.     

Human CD56+ Cytotoxic Lung Lymphocytes Kill Autologous Lung Cells in Chronic Obstructive Pulmonary Disease

CD56+ natural killer (NK) and CD56+ T cells, from sputum or bronchoalveolar lavage of subjects with chronic obstructive pulmonary disease (COPD) are more cytotoxic to highly susceptible NK targets than those from control subjects. Whether the same is true in lung parenchyma, and if NK activity actually contributes to emphysema progression are unknown. To address these questions, we performed two types of experiments on lung tissue from clinically-indicated resections (n = 60). First, we used flow cytometry on fresh single-cell suspension to measure expression of cell-surface molecules (CD56, CD16, CD8, NKG2D and NKp44) on lung lymphocytes and of the 6D4 epitope common to MICA and MICB on lung epithelial (CD326+) cells. Second, we sequentially isolated CD56+, CD8+ and CD4+ lung lymphocytes, co-cultured each with autologous lung target cells, then determined apoptosis of individual target cells using Annexin-V and 7-AAD staining. Lung NK cells (CD56+ CD3−) and CD56+ T cells (CD56+ CD3+) were present in a range of frequencies that did not differ significantly between smokers without COPD and subjects with COPD. Lung NK cells had a predominantly “cytotoxic” CD56+ CD16+ phenotype; their co-expression of CD8 was common, but the percentage expressing CD8 fell as FEV₁ % predicted decreased. Greater expression by autologous lung epithelial cells of the NKG2D ligands, MICA/MICB, but not expression by lung CD56+ cells of the activating receptor NKG2D, correlated inversely with FEV₁ % predicted. Lung CD56+ lymphocytes, but not CD4+ or CD8+ conventional lung T cells, rapidly killed autologous lung cells without additional stimulation. Such natural cytotoxicity was increased in subjects with severe COPD and was unexplained in multiple regression analysis by age or cancer as indication for surgery. These data show that as spirometry worsens in COPD, CD56+ lung lymphocytes exhibit spontaneous cytotoxicity of autologous structural lung cells, supporting their potential role in emphysema progression.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00281229","term_id":"NCT00281229"}}NCT00281229     

Deubiquitylation Machinery Is Required for Embryonic Polarity in Caenorhabditis elegans

The Caenorhabditis elegans one-cell embryo polarizes in response to a cue from the paternally donated centrosome and asymmetrically segregates cell fate determinants that direct the developmental program of the worm. We have found that genes encoding putative deubiquitylating enzymes (DUBs) are required for polarization of one-cell embryos. Maternal loss of the proteins MATH-33 and USP-47 leads to variable inability to correctly establish and maintain asymmetry as defined by posterior and anterior polarity proteins PAR-2 and PAR-3. The first observable defect is variable positioning of the centrosome with respect to the cell cortex and the male pronucleus. The severity of the polarity defects correlates with distance of the centrosome from the cortex. Furthermore, polarity defects can be bypassed by mutations that bring the centrosome in close proximity to the cortex. In addition we find that polarity and centrosome positioning defects can be suppressed by compromising protein turnover. We propose that the DUB activity of MATH-33 and USP-47 stabilizes one or more proteins required for association of the centrosome with the cortex. Because these DUBs are homologous to two members of a group of DUBs that act in fission yeast polarity, we tested additional members of that family and found that another C. elegans DUB gene, usp-46, also contributes to polarity. Our finding that deubiquitylating enzymes required for polarity in Schizosaccharomyces pombe are also required in C. elegans raises the possibility that these DUBs act through an evolutionarily conserved mechanism to control cell polarity.