A survey of new temperature-sensitive, embryonic-lethal mutations in C. elegans: 24 alleles of thirteen genes

To study essential maternal gene requirements in the early C. elegans embryo, we have screened for temperature-sensitive, embryonic lethal mutations in an effort to bypass essential zygotic requirements for such genes during larval and adult germline development. With conditional alleles, multiple essential requirements can be examined by shifting at different times from the permissive temperature of 15°C to the restrictive temperature of 26°C. Here we describe 24 conditional mutations that affect 13 different loci and report the identity of the gene mutations responsible for the conditional lethality in 22 of the mutants. All but four are mis-sense mutations, with two mutations affecting splice sites, another creating an in-frame deletion, and one creating a premature stop codon. Almost all of the mis-sense mutations affect residues conserved in orthologs, and thus may be useful for engineering conditional mutations in other organisms. We find that 62% of the mutants display additional phenotypes when shifted to the restrictive temperature as L1 larvae, in addition to causing embryonic lethality after L4 upshifts. Remarkably, we also found that 13 out of the 24 mutations appear to be fast-acting, making them particularly useful for careful dissection of multiple essential requirements. Our findings highlight the value of C. elegans for identifying useful temperature-sensitive mutations in essential genes, and provide new insights into the requirements for some of the affected loci.     

Composition and acidification of the culture medium influences chronological aging similarly in vineyard and laboratory yeast

Chronological aging has been studied extensively in laboratory yeast by culturing cells into stationary phase in synthetic complete medium with 2% glucose as the carbon source. During this process, acidification of the culture medium occurs due to secretion of organic acids, including acetic acid, which limits survival of yeast cells. Dietary restriction or buffering the medium to pH 6 prevents acidification and increases chronological life span. Here we set out to determine whether these effects are specific to laboratory-derived yeast by testing the chronological aging properties of the vineyard yeast strain RM11. Similar to the laboratory strain BY4743 and its haploid derivatives, RM11 and its haploid derivatives displayed increased chronological life span from dietary restriction, buffering the pH of the culture medium, or aging in rich medium. RM11 and BY4743 also displayed generally similar aging and growth characteristics when cultured in a variety of different carbon sources. These data support the idea that mechanisms of chronological aging are similar in both the laboratory and vineyard strains.     

Down-regulation of Tripartite-motif containing 22 expression in breast cancer is associated with a lack of p53-mediated induction

Tripartite-motif containing 22 (TRIM22) is a direct p53 target gene and inhibits the clonogenic growth of leukemic cells. Its expression in Wilms tumors is negatively associated with disease relapse. This study addresses if TRIM22 expression is de-regulated in breast carcinoma. Western blotting analysis of a panel of 10 breast cancer cell lines and 3 non-malignant mammary epithelial cell lines with a well-characterized TRIM22 monoclonal antibody showed that TRIM22 protein is greatly under-expressed in breast cancer cells as compared to non-malignant cell lines. Similarly, TRIM22 protein is significantly down-regulated in breast tumors as compared to matched normal breast tissues. Study of cell lines with methylation inhibitor and bisulfite sequencing indicates that TRIM22 promoter hypermethylation may not be the cause for TRIM22 under-expression in breast cancer. Instead, we found that TRIM22 protein level correlates strongly (R = 0.79) with p53 protein level in normal breast tissue, but this correlation is markedly impaired (R = 0.48) in breast cancer tissue, suggesting that there is some defects in p53 regulation of TRIM22 gene in breast cancer. This notion is supported by cell line studies, which showed that TRIM22 was no longer inducible by p53-activating genotoxic drugs in breast cancer cell lines and in a p53 null cell line H1299 transfected with wild type p53. In conclusion, this study shows that TRIM22 is greatly under-expressed in breast cancer. p53 dysfunction may be one of the mechanisms for TRIM22 down-regulation.     

The formins Cdc12 and For3 cooperate during contractile ring assembly in cytokinesis

Both de novo–assembled actin filaments at the division site and existing filaments recruited by directional cortical transport contribute to contractile ring formation during cytokinesis. However, it is unknown which source is more important. Here, we show that fission yeast formin For3 is responsible for node condensation into clumps in the absence of formin Cdc12. For3 localization at the division site depended on the F-BAR protein Cdc15, and for3 deletion was synthetic lethal with mutations that cause defects in contractile ring formation. For3 became essential in cells expressing N-terminal truncations of Cdc12, which were more active in actin assembly but depended on actin filaments for localization to the division site. In tetrad fluorescence microscopy, double mutants of for3 deletion and cdc12 truncations were severely defective in contractile ring assembly and constriction, although cortical transport of actin filaments was normal. Together, these data indicate that different formins cooperate in cytokinesis and that de novo actin assembly at the division site is predominant for contractile ring formation.     

Five chemically rich species of tropical marine cyanobacteria of the genus Okeania gen. nov. (Oscillatoriales,Cyanoprokaryota)

An adverse consequence of applying morphology‐based taxonomic systems to catalog cyanobacteria, which generally are limited in the number of available morphological characters, is a fundamental underestimation of natural biodiversity. In this study, we further dissect the polyphyletic cyanobacterial genus Lyngbya and delineate the new genus Okeania gen. nov. Okeania is a tropical and subtropical, globally distributed marine group abundant in the shallow‐water benthos. Members of Okeania are of considerable ecological and biomedical importance because specimens within this group biosynthesize biologically active secondary metabolites and are known to form blooms in coastal benthic environments. Herein, we describe five species of the genus Okeania: O. hirsuta (type species of the genus), O. plumata, O. lorea, O. erythroflocculosa, and O. comitata, under the provisions of the International Code of Nomenclature for Algae, Fungi, and Plants. All five Okeania species were morphologically, phylogenetically, and chemically distinct. This investigation provides a classification system that is able to identify Okeania spp. and predict their production of bioactive secondary metabolites.     

The temporal development and additivity of plant-soil feedback in perennial grasses

Background

Current knowledge of plant-soil feedback is based largely on single end point studies with soils conditioned by monocultures, but accounting for variability in the ecological impacts of feedback effects may require understanding how feedback develops over time and in multi-species plant communities.

Methods

To examine temporal development and additivity of feedback, two pairs of native and non-native congeneric grasses were grown alone or in mixtures to create six soil conditioning treatments. We measured plant growth and feedback on the soils over 19 months and addressed whether plant biomass was additive or non-additive between soils treated by mixtures and their constituent monocultures.

Results

For native grasses, plant-soil feedback either became progressively more negative through time or switched from neutral to negative. Feedback to non-native grasses was variably neutral to positive. Final biomass of the grasses growing on soils conditioned by mixtures was generally an additive function of growth on soils conditioned by the component monocultures, except native grasses growing in soils conditioned by their own congener mixtures, which were non-additive.

Conclusions

Temporal variation and non-additivity in feedback suggest that extrapolation to communities may be complex. More work is needed to assess the generality of temporal and scaling effects.     

Genetic structure and diversity of coffee (Coffea) across Africa and the Indian Ocean islands revealed using microsatellites

Background and Aims

The coffee genus (Coffea) comprises 124 species, and is indigenous to the Old World Tropics. Due to its immense economic importance, Coffea has been the focus of numerous genetic diversity studies, but despite this effort it remains insufficiently studied. In this study the genetic diversity and genetic structure of Coffea across Africa and the Indian Ocean islands is investigated.

Methods

Genetic data were produced using 13 polymorphic nuclear microsatellite markers (simple sequence repeats, SSRs), including seven expressed sequence tag-SSRs, and the data were analysed using model- and non-model-based methods. The study includes a total of 728 individuals from 60 species.

Key Results

Across Africa and the Indian Ocean islands Coffea comprises a closely related group of species with an overall pattern of genotypes running from west to east. Genetic structure was identified in accordance with pre-determined geographical regions and phylogenetic groups. There is a good relationship between morpho-taxonomic species delimitations and genetic units. Genetic diversity in African and Indian Ocean Coffea is high in terms of number of alleles detected, and Madagascar appears to represent a place of significant diversification in terms of allelic richness and species diversity.

Conclusions

Cross-species SSR transferability in African and Indian Ocean islands Coffea was very efficient. On the basis of the number of private alleles, diversification in East Africa and the Indian Ocean islands appears to be more recent than in West and West-Central Africa, although this general trend is complicated in Africa by the position of species belonging to lineages connecting the main geographical regions. The general pattern of phylogeography is not in agreement with an overall east to west (Mascarene, Madagascar, East Africa, West Africa) increase in genome size, the high proportion of shared alleles between the four regions or the high numbers of exclusive shared alleles between pairs or triplets of regions.     

Changes to soil organic N dynamics with leguminous woody plant encroachment into grasslands

Encroachment of nitrogen-fixing trees and shrubs into grasslands and savannas is a well-documented land cover change that occurs worldwide. In the Rio Grande Plains region of southern Texas, previous studies have shown woody encroachment by leguminous Prosopis glandulosa (mesquite) trees increases soil C and N, decreases microbial biomass N relative to soil N, and accelerates N mineralization and nitrification. We examined responses of the dominant organic N components in soil (amino acids and amino sugars) and two soil-bound protein-N acquiring enzymes (arylamidase and β-N-acetylglucosaminidase) along a grassland-to-woodland successional chronosequence to determine changes to soil N chemistry and extractability. The proportion of total N held within amino compounds was significantly lower in the woodlands (47 %) relative to the grassland soils (62 %). This increase in non-hydrolysable N was accompanied by increases in plant cell wall derived amino acids (e.g. hydroxyproline, serine) and losses of microbial amino sugars, indicating the woodland organic N pool was altered in composition and potentially in quality, either because it was more structurally protected or difficult to degrade due to polymerization/condensation reactions. Soil carbon-normalized activities of both soil-bound N-acquiring enzymes were significantly higher in woodland soils, consistent with changes in the biochemical composition of organic N. Although soil total N increases following woody encroachment, this additional organic N appears to be less extractable by chemical hydrolysis and thus potentially in more refractory forms, which may limit microbial N accessibility, slow the cycling of soil organic carbon, and contribute to observed soil C and N accrual in these systems.     

Biotechnological advances on Penicillin G acylase: Pharmaceutical implications,unique expression mechanism and production strategies

In light of unrestricted use of first-generation penicillins, these antibiotics are now superseded by their semisynthetic counterparts for augmented antibiosis. Traditional penicillin chemistry involves the use of hazardous chemicals and harsh reaction conditions for the production of semisynthetic derivatives and, therefore, is being displaced by the biosynthetic platform using enzymatic transformations. Penicillin G acylase (PGA) is one of the most relevant and widely used biocatalysts for the industrial production of β-lactam semisynthetic antibiotics. Accordingly, considerable genetic and biochemical engineering strategies have been devoted towards PGA applications. This article provides a state-of-the-art review in recent biotechnological advances associated with PGA, particularly in the production technologies with an emphasis on using the Escherichia coli expression platform.     

Mass spectrometry analysis of oxidized phosphatidylcholine and phosphatidylethanolamine

Oxidized phospholipids (OxPLs) are rapidly becoming recognized as important mediators of cellular and immune signaling. They are generated either enzymatically or non-enzymatically and 100s of structures exist of which only a small fraction have been analyzed to date. Pleiotropic activities, including regulation of adhesion molecule expression, pro-coagulant activity and inhibition of Toll-like receptor signaling have been observed and some are detected in models of human and animal disease, including atherosclerosis and infection. More recently, the acute generation of specific oxidized phospholipids by cellular enzymes in immune cells was reported. Assays for analysis and quantification of OxPLs were first developed approx 15years ago, primarily for hydro(pero)xy-species. Many were based on monitoring a single precursor ion with/without LC separation, based on the PL headgroup. Others combined LC with monitoring precursor to product transitions, but were unable to provide information regarding position of oxidation on unsaturated sn-2 fatty acid due to sensitivity issues. More recently, LC/MS/MS methods for specific OxPLs have been reported that enable high sensitivity quantitation in biological samples. In this review, widely used methods for detecting and quantifying various classes of OxPL will be summarized, along with practical advice for their use. In particular, the focus will be on LC/MS/MS, which today is almost universally the method of choice.