We developed a procedure for automated confocal microscopy to image the effect of the non-essential yeast gene deletion set on the localisation of the plasma membrane GFP-labelled protein Mrh1p-GFP. To achieve this it was necessary to devise an expression system expressing Redstar2 RFP-fluorescence specifically in the nucleus, mCherry RFP at a lower intensity in the cytoplasm and Mrh1p-GFP in the plasma membrane. This fluorescence labelling scheme utilising specifically designed image analysis scripts allowed automated segmentation of the cells into sub-regions comprising nuclei, cytoplasm and cell-surface. From this high-throughput high content screening approach we were able to determine that gene deletions including emc1Δ, emc2Δ, emc3Δ, emc4Δ, emc5Δ and emc6Δ, caused intracellular mislocalisation at the ER of a plasma membrane protein Mrh1p-GFP. CPY processing patterns were unaffected in these mutants and collectively our data suggest a transport role for the EMC genes within the early secretory pathway. HAC1 is central to the unfolded protein response (UPR) and in its absence, i.e. the absence of UPR, emc1Δ-, emc3Δ-, emc4Δ-, emc5Δ-hac1Δ double mutants were specifically hypersensitive to ER-stress (tunicamycin) lending credence to the usefulness of the high content microscope screening for discovery of functional effects of single mutants.

Staphylococcus aureus remains a causative agent for morbidity and mortality worldwide. This is in part a result of antimicrobial resistance, highlighting the need to uncover novel antibiotic targets and to discover new therapeutic agents. In the present study, we explored the possibility that iron-sulfur (Fe-S) cluster synthesis is a viable antimicrobial target. RNA interference studies established that Suf (sulfur mobilization)-dependent Fe-S cluster synthesis is essential in S. aureus. We found that sufCDSUB were cotranscribed and that suf transcription was positively influenced by sigma factor B. We characterized an S. aureus strain that contained a transposon inserted in the intergenic space between sufC and sufD (sufD*), resulting in decreased transcription of sufSUB. Consistent with the transcriptional data, the sufD* strain had multiple phenotypes associated with impaired Fe-S protein maturation. They included decreased activities of Fe-S cluster-dependent enzymes, decreased growth in media lacking metabolites that require Fe-S proteins for synthesis, and decreased flux through the tricarboxylic acid (TCA) cycle. Decreased Fe-S cluster synthesis resulted in sensitivity to reactive oxygen and reactive nitrogen species, as well as increased DNA damage and impaired DNA repair. The sufD* strain also exhibited perturbed intracellular nonchelated Fe pools. Importantly, the sufD* strain did not exhibit altered exoprotein production or altered biofilm formation, but it was attenuated for survival upon challenge by human polymorphonuclear leukocytes. The results presented are consistent with the hypothesis that Fe-S cluster synthesis is a viable target for antimicrobial development.KEYWORDS iron, sulfur, cluster, Staphylococcus aureus, Suf, neutrophil S taphylococcus aureus is a human commensal that causes morbidity and mortality worldwide. While it is responsible for low-morbidity maladies, such as folliculitis, it is also capable of causing fatal afflictions, such as endocarditis, bacteremia, and toxic shock syndrome (1, 2). Bacterial antibiotic resistance continues to increase and to be problematic. Infections caused by antibiotic-resistant S. aureus result in increased mortality, increased stress on the health care system, and an increased financial burden (3, 4). Current FDA-approved antibacterials target a limited number of metabolic processes (5). Developing antibacterials that target alternate processes would expand treatment options and aid in multidrug therapy. These facts highlight the need for

Mammographic breast density (MBD) has been proven to be an important risk factor for breast cancer and an important determinant of mammographic screening performance. The measurement of density has changed dramatically since its inception. Initial qualitative measurement methods have been found to have limited consistency between readers, and in regards to breast cancer risk. Following the introduction of full-field digital mammography, more sophisticated measurement methodology is now possible. Automated computer-based density measurements can provide consistent, reproducible, and objective results. In this review paper, we describe various methods currently available to assess MBD, and provide a discussion on the clinical utility of such methods for breast cancer screening.

Eukaryotic genetic interaction networks (GINs) are extensively described in the Saccharomyces cerevisiae S288C model using deletion libraries, yet being limited to this one genetic background, not informative to individual drug response. Here we created deletion libraries in three additional genetic backgrounds. Statin response was probed with five queries against four genetic backgrounds. The 20 resultant GINs representing drug–gene and gene–gene interactions were not conserved by functional enrichment, hierarchical clustering, and topology-based community partitioning. An unfolded protein response (UPR) community exhibited genetic background variation including different betweenness genes that were network bottlenecks, and we experimentally validated this UPR community via measurements of the UPR that were differentially activated and regulated in statin-resistant strains relative to the statin-sensitive S288C background. These network analyses by topology and function provide insight into the complexity of drug response influenced by genetic background.

Summary S. aureus SufT is composed solely of the domain of unknown function 59 (DUF59) and has a role in the maturation of iron-sulfur (Fe-S) proteins. We report that SufT is essential for S. aureus when growth is heavily reliant upon lipoamide-utilizing enzymes, but dispensable when this reliance is decreased. LipA requires Fe-S clusters for lipoic acid (LA) synthesis and a ΔsufT strain had phenotypes suggestive of decreased LA production and decreased activities of lipoamide-requiring enzymes. Fermentative growth, a null clpC allele, or decreased flux through the TCA cycle diminished the demand for LA and rendered SufT non-essential. Abundance of the Fe-S cluster carrier Nfu was increased in a ΔclpC strain and a null clpC allele was unable to suppress the LA requirement of a ΔsufT Δnfu strain. Over-expression of nfu suppressed the LA requirement of the ΔsufT strain. We propose a model wherein SufT, and by extension the DUF59, is essential for the maturation of holo-LipA in S. aureus cells experiencing a high demand for lipoamide-dependent enzymes. The findings presented suggest that the demand for products of Fe-S enzymes is a factor governing the usage of one Fe-S cluster assembly factor over another in the maturation of apo-proteins.