Effects of deletion mutations in the yeast Ces1 protein on cell growth and morphology and on high copy suppression of mutations in mRNA capping enzyme and translation initiation factor 4A

The homologous Saccharomyces cerevisiae genes CES1 and CES4 act as high copy suppressors of temperature-sensitive mutations of Ceg1p, the yeast mRNA capping enzyme. Neither CES1 nor CES4 is essential for cell growth. We find that a double deletion mutant (Δces1 Δces4) grows at 25-37°C, but not at 16°C. Δces1 Δces4 cells display gross defects in cell shape and budding even at permissive temperatures. Functional analysis of CES1 deletion mutants defines a 145 amino acid C-terminal segment of the 915 amino acid Ces1 protein that is necessary and sufficient to complement the Δces1 Δces4 cold-sensitive phenotype, to restore normal morphology and to suppress the temparature-sensitive mutant ceg1-25. A 147 amino acid C-terminal segment of the 942 amino acid Ces4 protein is sufficient to carry out these same functions. Within this carboxyl domain Ces1p and Ces4p are 80% identical to one another. We report isolation of CES1 in a separate screen for high copy suppression of a temperature-sensitive mutation (A79V) of the yeast translation initiation factor Tif1p (eIF-4A). Deletion of the N-terminal 249 amino acids of Ces1p abolished tif1-A79V suppressor function. CES4 on a multicopy plasmid was unable to suppress tif1-A79V. We surmise that whereas the carboxyl domains of Ces1p and Ces4p are functionally redundant in controlling cell morphology and in suppressing ceg1-25, full-length Ces1p and Ces4p evince distinct genetic interactions that are likely mediated by their N-terminal segment

Anthropocentric perspective of production before and within Industry 4.0

Abstract This paper presents a systematic literature review (SLR) of the anthropocentric perspective of production before and after (or, better, within) Industry 4.0. We identify central research clusters regarding traditional Anthropocentric Production Systems (APS) and Anthropocentric Cyber Physical Production Systems. By comparing the two perspectives, we are able to analyse new emerging paradigms in anthropocentric production caused by Industry 4.0. We further make prediction of the future role of the human operator, his needed knowledge and capabilities and how assistance systems support the Operator 4.0. Our paper gives a brief outlook of current and needed future research. It builds grounds for further scholarly discussion on the role of humans in the factory of the future

Characterization of the ATPase and unwinding activities of the yeast DEAD-box protein Has1p and the analysis of the roles of the conserved motifs

The yeast DEAD-box protein Has1p is required for the maturation of 18S rRNA, the biogenesis of 40S r-subunits and for the processing of 27S pre-rRNAs during 60S r-subunit biogenesis. We purified recombinant Has1p and characterized its biochemical activities. We show that Has1p is an RNA-dependent ATPase in vitro and that it is able to unwind RNA/DNA duplexes in an ATP-dependent manner. We also report a mutational analysis of the conserved residues in motif I (86AKTGSGKT93), motif III (228SAT230) and motif VI (375HRVGRTARG383). The in vivo lethal K92A substitution in motif I abolishes ATPase activity in vitro. The mutations S228A and T230A partially dissociate ATPase and helicase activities, and they have cold-sensitive and lethal growth phenotypes, respectively. The H375E substitution in motif VI significantly decreased helicase but not ATPase activity and was lethal in vivo. These results suggest that both ATPase and unwinding activities are required in vivo. Has1p possesses a Walker A-like motif downstream of motif VI (383GTKGKGKS390). K389A substitution in this motif significantly increases the Has1p activity in vitro, which indicates it potentially plays a role as a negative regulator. Finally, rRNAs and poly(A) RNA serve as the best stimulators of the ATPase activity of Has1p among the tested RNA

Characterization of the ATPase and unwinding activities of the yeast DEAD-box protein Has1p and the analysis of the roles of the conserved motifs

The yeast DEAD-box protein Has1p is required for the maturation of 18S rRNA, the biogenesis of 40S r-subunits and for the processing of 27S pre-rRNAs during 60S r-subunit biogenesis. We purified recombinant Has1p and characterized its biochemical activities. We show that Has1p is an RNA-dependent ATPase in vitro and that it is able to unwind RNA/DNA duplexes in an ATP-dependent manner. We also report a mutational analysis of the conserved residues in motif I ((86)AKTGSGKT(93)), motif III ((228)SAT(230)) and motif VI ((375)HRVGRTARG(383)). The in vivo lethal K92A substitution in motif I abolishes ATPase activity in vitro. The mutations S228A and T230A partially dissociate ATPase and helicase activities, and they have cold-sensitive and lethal growth phenotypes, respectively. The H375E substitution in motif VI significantly decreased helicase but not ATPase activity and was lethal in vivo. These results suggest that both ATPase and unwinding activities are required in vivo. Has1p possesses a Walker A-like motif downstream of motif VI ((383)GTKGKGKS(390)). K389A substitution in this motif significantly increases the Has1p activity in vitro, which indicates it potentially plays a role as a negative regulator. Finally, rRNAs and poly(A) RNA serve as the best stimulators of the ATPase activity of Has1p among the tested RNAs

Visit complexity, diagnostic uncertainty, and antibiotic prescribing for acute cough in primary care: a retrospective study

Background: Guidelines and performance measures recommend avoiding antibiotics for acute cough/acute bronchitis and presume visits are straightforward with simple diagnostic decision-making. We evaluated clinician-assigned diagnoses, diagnostic uncertainty, and antibiotic prescribing for acute cough visits in primary care. Methods: We conducted a retrospective analysis of acute cough visits – cough lasting ≤21 days in adults 18–64 years old without chronic lung disease – in a primary care practice from March 2011 through June 2012. Results: Of 56,301 visits, 962 (2%) were for acute cough. Clinicians diagnosed patients with 1, 2, or ≥ 3 cough-related diagnoses in 54%, 35%, and 11% of visits, respectively. The most common principal diagnoses were upper respiratory infection (46%), sinusitis (10%), acute bronchitis (9%), and pneumonia (8%). Clinicians prescribed antibiotics in 22% of all visits: 65% of visits with antibiotic-appropriate diagnoses and 4% of visits with non-antibiotic-appropriate diagnoses. Clinicians expressed diagnostic uncertainty in 16% of all visits: 43% of visits with antibiotic-appropriate diagnoses and 5% of visits with non-antibiotic-appropriate diagnoses. Clinicians expressed uncertainty more often when prescribing antibiotics than when not prescribing antibiotics (30% vs. 12%; p < 0.001). As the number of visit diagnoses increased from 1 to 2 to ≥ 3, clinicians were more likely to express diagnostic uncertainty (5%, 25%, 40%, respectively; p < 0.001) and prescribe antibiotics (16%, 25%, 41%, respectively; p < 0.001). Conclusions: Acute cough may be more complex and have more diagnostic uncertainty than guidelines and performance measures presume. Efforts to reduce antibiotic prescribing for acute cough should address diagnostic complexity and uncertainty that clinicians face

A type IV modification-dependent restriction enzyme SauUSI from Staphylococcus aureus subsp. aureus USA300

A gene encoding a putative DNA helicase from Staphylococcus aureus USA300 was cloned and expressed in Escherichia coli. The protein was purified to over 90% purity by chromatography. The purified enzyme, SauUSI, predominantly cleaves modified DNA containing 5mC and 5-hydroxymethylcytosine. Cleavage of 5mC-modified plasmids indicated that the sites S5mCNGS (S = C or G) are preferentially digested. The endonuclease activity requires the presence of adenosine triphosphate (ATP) or dATP whereas the non-hydrolyzable γ-S-ATP does not support activity. SauUSI activity was inhibited by ethylenediaminetetraacetic acid. It is most active in Mg++ buffers. No companion methylase gene was found near the SauUSI restriction gene. The absence of a cognate methylase and cleavage of modified DNA indicate that SauUSI belongs to type IV restriction endonucleases, a group that includes EcoK McrBC and Mrr. SauUSI belongs to a family of highly similar homologs found in other sequenced S. aureus, S. epidermidis and S. carnosus genomes. More distant SauUSI orthologs can be found in over 150 sequenced bacterial/archaea genomes. Finally, we demonstrated the biological function of the type IV REase in restricting 5mC-modified plasmid DNA by transformation into clinical S. aureus strain SA564, and in restricting phage λ infection when the endonuclease is expressed in E. col

BiOutils: an interface to connect university laboratories with microbiology classes in schools

The contribution of microbiology to the scientific advances of modern experimental biology has very often made the difference. Despite this, its role as an independent discipline has slowly started to fade away. This situation has been worsening due to (i) a marginal role of microbiology in academic curricula and (ii) a low or misplaced interest by the public at large towards this field of study. In order to counter this phenomenon, microbiology researchers and passionate scientists have made several efforts to engage and inform the broad public and academic policymakers about the importance of microbiology as an independent discipline. One of the approaches used in this direction is to support the teaching of microbiology in schools. BiOutils, a science communication platform based within a microbiology lab, has been committed to this goal since its creation in 2007. In this article, we describe how the platform is able to work in synergy with school teachers, providing engaging activities that can be performed in schools' classrooms. Our aim is to provide a perspective on how every microbiology lab with little costs and efforts can support the teaching of a discipline that will remain independent thanks to the fascination that they will be able to transmi

Primary care clinicians’ perceptions about antibiotic prescribing for acute bronchitis: a qualitative study

Background: Clinicians prescribe antibiotics to over 65% of adults with acute bronchitis despite guidelines stating that antibiotics are not indicated. Methods: To identify and understand primary care clinician perceptions about antibiotic prescribing for acute bronchitis, we conducted semi-structured interviews with 13 primary care clinicians in Boston, Massachusetts and used thematic content analysis. Results: All the participants agreed with guidelines that antibiotics are not indicated for acute bronchitis and felt that clinicians other than themselves were responsible for overprescribing. Barriers to guideline adherence included 6 themes: (1) perceived patient demand, which was the main barrier, although some clinicians perceived a recent decrease; (2) lack of accountability for antibiotic prescribing; (3) saving time and money; (4) other clinicians’ misconceptions about acute bronchitis; (5) diagnostic uncertainty; and (6) clinician dissatisfaction in failing to meet patient expectations. Strategies to decrease inappropriate antibiotic prescribing included 5 themes: (1) patient educational materials; (2) quality reporting; (3) clinical decision support; (4) use of an over-the-counter prescription pad; and (5) pre-visit triage and education by nurses to prevent visits. Conclusions: Clinicians continued to cite patient demand as the main reason for antibiotic prescribing for acute bronchitis, though some clinicians perceived a recent decrease. Clinicians felt that other clinicians were responsible for inappropriate antibiotic prescribing and that better pre-visit triage by nurses could prevent visits and change patients’ expectations. Electronic supplementary material The online version of this article (doi:10.1186/s12875-014-0194-5) contains supplementary material, which is available to authorized users

Membrane stress is coupled to a rapid translational control of gene expression in chlorpromazine-treated cells

Chlorpromazine (CPZ) is a small permeable cationic amphiphilic molecule that inserts into membrane bilayers and binds to anionic lipids such as poly-phosphoinositides (PIs). Since PIs play important roles in many cellular processes, including signaling and membrane trafficking pathways, it has been proposed that CPZ affects cellular growth functions by preventing the recruitment of proteins with specific PI-binding domains. In this study, we have investigated the biological effects of CPZ in the yeast Saccharomyces cerevisiae. We screened a collection of approximately 4,800 gene knockout mutants, and found that mutants defective in membrane trafficking between the late-Golgi and endosomal compartments are highly sensitive to CPZ. Microscopy and transport analyses revealed that CPZ affects membrane structure of organelles, blocks membrane transport and activates the unfolded protein response (UPR). In addition, CPZ-treatment induces phosphorylation of the translation initiation factor (eIF2α), which reduces the general rate of protein synthesis and stimulates the production of Gcn4p, a major transcription factor that is activated in response to environmental stresses. Altogether, our results reveal that membrane stress within the cells rapidly activates an important gene expression program, which is followed by a general inhibition of protein synthesis. Remarkably, the increase of phosphorylated eIF2α and protein synthesis inhibition were also detected in CPZ-treated NIH-3T3 fibroblasts, suggesting the existence of a conserved mechanism of translational regulation that operates during a membrane stres