The Cul4-Ddb1(Cdt)² ubiquitin ligase inhibits invasion of a boundary-associated antisilencing factor into heterochromatin

Partitioning of chromosomes into euchromatic and heterochromatic domains requires mechanisms that specify boundaries. The S. pombe JmjC family protein Epe1 prevents the ectopic spread of heterochromatin and is itself concentrated at boundaries. Paradoxically, Epe1 is recruited to heterochromatin by HP1 silencing factors that are distributed throughout heterochromatin. We demonstrate here that the selective enrichment of Epe1 at boundaries requires its regulation by the conserved Cul4-Ddb1(Cdt)2 ubiquitin ligase, which directly recognizes Epe1 and promotes its polyubiquitylation and degradation. Strikingly, in cells lacking the ligase, Epe1 persists in the body of heterochromatin thereby inducing a defect in gene silencing. Epe1 is the sole target of the Cul4-Ddb1(Cdt)2 complex whose destruction is necessary for the preservation of heterochromatin. This mechanism acts parallel with phosphorylation of HP1/Swi6 by CK2 to restrict Epe1. We conclude that the ubiquitin-dependent sculpting of the chromosomal distribution of an antisilencing factor is critical for heterochromatin boundaries to form correctly.     

A chromogenic assay of substrate depletion by thiol dioxygenases

A fast and easy method for enzyme activity assays using the chromogenic Ellman reagent, 5,5′-dithiobis(2-nitrobenzoic acid), was developed. The method was used to measure the activity of the nonheme mono-iron enzyme cysteine dioxygenase. Quantifying the depletion of the substrate, cysteine, allowed standard kinetic parameters to be determined for the enzyme from Rattus norvegicus. The assay was also used to quickly test the effects of ionic strength, pH, enzyme storage conditions, and potential inhibitors and activators. This assay facilitates a higher throughput than available HPLC-based assays, as it enjoys the advantages of fewer sample handling steps, implementation in a 96-well format, and speed. In addition, the relative specificity of Ellman’s reagent, coupled with its reaction with a wide range of thiols, means that this assay is applicable to many enzymes. Finally, the use of readily available reagents and instrumentation means that this assay can be used by practically any research group to compare results with those of other groups.     

Error propagation methods for LCA—a comparison

Purpose

The analysis of uncertainty in life cycle assessment (LCA) studies has been a topic for more than 10 years, and many commercial LCA programs now feature a sampling approach called Monte Carlo analysis. Yet, a full Monte Carlo analysis of a large LCA system, for instance containing the 4,000 unit processes of ecoinvent v2.2, is rarely carried out by LCA practitioners. One reason for this is computation time. An alternative faster than Monte Carlo method is analytical error propagation by means of a Taylor series expansion; however, this approach suffers from being explained in the literature in conflicting ways, hampering implementation in most software packages for LCA. The purpose of this paper is to compare the two different approaches from a theoretical and practical perspective.

Methods

In this paper, we compare the analytical and sampling approaches in terms of their theoretical background and their mathematical formulation. Using three case studies—one stylized, one real-sized, and one input–output (IO)-based—we approach these techniques from a practical perspective and compare them in terms of speed and results.

Results

Depending on the precise question, a sampling or an analytical approach provides more useful information. Whenever they provide the same indicators, an analytical approach is much faster but less reliable when the uncertainties are large.

Conclusions

For a good analysis, analytical and sampling approaches are equally important, and we recommend practitioners to use both whenever available, and we recommend software suppliers to implement both.     

Changes in Colorectal Carcinoma Genomes under Anti-EGFR Therapy Identified by Whole-Genome Plasma DNA Sequencing

Monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR), such as cetuximab and panitumumab, have evolved to important therapeutic options in metastatic colorectal cancer (CRC). However, almost all patients with clinical response to anti-EGFR therapies show disease progression within a few months and little is known about mechanism and timing of resistance evolution. Here we analyzed plasma DNA from ten patients treated with anti-EGFR therapy by whole genome sequencing (plasma-Seq) and ultra-sensitive deep sequencing of genes associated with resistance to anti-EGFR treatment such as KRAS, BRAF, PIK3CA, and EGFR. Surprisingly, we observed that the development of resistance to anti-EGFR therapies was associated with acquired gains of KRAS in four patients (40%), which occurred either as novel focal amplifications (n = 3) or as high level polysomy of 12p (n = 1). In addition, we observed focal amplifications of other genes recently shown to be involved in acquired resistance to anti-EGFR therapies, such as MET (n = 2) and ERBB2 (n = 1). Overrepresentation of the EGFR gene was associated with a good initial anti-EGFR efficacy. Overall, we identified predictive biomarkers associated with anti-EGFR efficacy in seven patients (70%), which correlated well with treatment response. In contrast, ultra-sensitive deep sequencing of KRAS, BRAF, PIK3CA, and EGFR did not reveal the occurrence of novel, acquired mutations. Thus, plasma-Seq enables the identification of novel mutant clones and may therefore facilitate early adjustments of therapies that may delay or prevent disease progression.     

Occupational Exposure to Ultrafine Particles among Airport Employees - Combining Personal Monitoring and Global Positioning System

Background

Exposure to ultrafine particles (UFP) has been linked to cardiovascular and lung diseases. Combustion of jet fuel and diesel powered handling equipment emit UFP resulting in potentially high exposure levels among employees working at airports. High levels of UFP have been reported at several airports, especially on the apron, but knowledge on individual exposure profiles among different occupational groups working at an airport is lacking.

Purpose

The aim of this study was to compare personal exposure to UFP among five different occupational groups working at Copenhagen Airport (CPH).

Method

30 employees from five different occupational groups (baggage handlers, catering drivers, cleaning staff and airside and landside security) at CPH were instructed to wear a personal monitor of particle number concentration in real time and a GPS device. The measurements were carried out on 8 days distributed over two weeks in October 2012. The overall differences between the groups were assessed using linear mixed model.

Results

Data showed significant differences in exposure levels among the groups when adjusted for variation within individuals and for effect of time and date (p<0.01). Baggage handlers were exposed to 7 times higher average concentrations (geometric mean, GM: 37×10³ UFP/cm³, 95% CI: 25–55×10³ UFP/cm³) than employees mainly working indoors (GM: 5×10³ UFP/cm³, 95% CI: 2–11×10³ UFP/cm³). Furthermore, catering drivers, cleaning staff and airside security were exposed to intermediate concentrations (GM: 12 to 20×10³ UFP/cm³).

Conclusion

The study demonstrates a strong gradient of exposure to UFP in ambient air across occupational groups of airport employees.     

CRYPTIC CHOICE OF CONSPECIFIC SPERM CONTROLLED BY THE IMPACT OF OVARIAN FLUID ON SPERM SWIMMING BEHAVIOR

Despite evidence that variation in male–female reproductive compatibility exists in many fertilization systems, identifying mechanisms of cryptic female choice at the gamete level has been a challenge. Here, under risks of genetic incompatibility through hybridization, we show how salmon and trout eggs promote fertilization by conspecific sperm. Using in vitro fertilization experiments that replicate the gametic microenvironment, we find complete interfertility between both species. However, if either species’ ova were presented with equivalent numbers of both sperm types, conspecific sperm gained fertilization precedence. Surprisingly, the species’ identity of the eggs did not explain this cryptic female choice, which instead was primarily controlled by conspecific ovarian fluid, a semiviscous, protein‐rich solution that bathes the eggs and is released at spawning. Video analyses revealed that ovarian fluid doubled sperm motile life span and straightened swimming trajectory, behaviors allowing chemoattraction up a concentration gradient. To confirm chemoattraction, cell migration tests through membranes containing pores that approximated to the egg micropyle showed that conspecific ovarian fluid attracted many more spermatozoa through the membrane, compared with heterospecific fluid or water. These combined findings together identify how cryptic female choice can evolve at the gamete level and promote reproductive isolation, mediated by a specific chemoattractive influence of ovarian fluid on sperm swimming behavior.     

A Material History of Australia

This article presents an analysis of the material history of Australia in the period 1975–2005. The values of economy‐wide indicators of material flow roughly trebled since 1975, and we identify the drivers of this change through structural decomposition analysis. The purpose of this work is to delve beneath the top‐level trends in material flow growth to investigate the structural changes in the economy that have been driving this growth. The major positive drivers of this change were the level of exports, export mix, industrial structure, affluence, and population. Only improvements in material intensity offered retardation of growth in material flow. Other structural components had only small effects at the aggregate level. At a more detailed level, however, the importance of the mineral sectors became apparent. Improvements in mining techniques have reduced material requirements, but increased consumption within the economy and increased exports have offset these reductions. The full roll out of material flow accounting through Australian society and business and a systematic response to its implications will require change in the national growth focus of the last two generations, with serious consideration needed to reverse the current volume‐focused growth of the economy and also to recast neoliberal and globalized trade policies that have dominated the globe for the past decades.     

SM-protein-controlled ER-associated degradation discriminates between different SNAREs

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a specialized activity of the ubiquitin-proteasome system that is involved in clearing the ER of aberrant proteins and regulating the levels of specific ER-resident proteins. Here we show that the yeast ER-SNARE Ufe1, a syntaxin (Qa-SNARE) involved in ER membrane fusion and retrograde transport from the Golgi to the ER, is prone to degradation by an ERAD-like mechanism. Notably, Ufe1 is protected against degradation through binding to Sly1, a known SNARE regulator of the Sec1-Munc18 (SM) protein family. This mechanism is specific for Ufe1, as the stability of another Sly1 partner, the Golgi Qa-SNARE Sed5, is not influenced by Sly1 interaction. Thus, our findings identify Sly1 as a discriminating regulator of SNARE levels and indicate that Sly1-controlled ERAD might regulate the balance between different Qa-SNARE proteins.