COMPUTATIONAL FLUID DYNAMICS ANALYSIS OF NOx REDUCTION BY AMMONIA INJECTION IN THE MAN B&W 7S50MC MARINE ENGINE

Taking into account the importance of NOx (nitrogen oxides) emissions from marine engines and the current increasingly restrictive legislation, this work aims to develop a numerical model to study NOx reduction. To this end, direct injection of NH3 (ammonia) into the combustion chamber was proposed in the MAN B&W 7S50MC marine engine. The numerical model was employed to analyze several injection temperatures, injection timings and ammonia to fuel ratios, obtaining NOx reductions of almost 60%. Besides, a comparison between ammonia injection and water injection was done. The results showed that ammonia is more efficient than water to reduce NOx with a negligible influence on other pollutants such as CO (carbon monoxide) and HC (hydrocarbons). Nevertheless, ammonia is efficient in a very restrictive temperature and injection timing range. This numerical model was compared with experimental measurements, obtaining satisfactory results which validate the work.

Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM)

Understanding how in eukaryotic cells thousands of proteins are sorted from each other through the secretory pathway and delivered to their correct destinations is a central issue of cell biology. We have further investigated in yeast how two distinct types of cargo proteins are sorted into different endoplasmic reticulum (ER) exit sites (ERES) for their differential ER export to the Golgi apparatus. We used an optimized protocol that combines a live cell dual-cargo ER export system with a 3D simultaneous multi-color high-resolution live cell microscopy called Super-resolution Confocal Live Imaging Microscopy (SCLIM). Here, we describe this protocol, which is based on the reversible ER retention of two de novo co-expressed cargos by blocking COPII function upon incubation of the thermo-sensitive COPII allele sec31-1 at restrictive temperature (37°C). ER export is restored by shifting down to permissive temperature (24°C) and progressive incorporation of the two different types of cargos into the fluorescently labelled ERES can be then simultaneously captured at 3D high spatial resolution by SCLIM microscopy. By using this protocol, we have shown that newly synthesized glycosylphosphatidylinositol (GPI)-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized ERES that are distinct from those used by transmembrane secretory proteins. Furthermore, we showed that the chain length of the ceramide present in the ER membrane is critical for this sorting selectivity. Therefore, thanks to the presented method we could obtain the first direct in vivo evidence for lipid chain length-based protein cargo sorting into selective ERES.

Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by Super-resolution Confocal Live Imaging Microscopy (SCLIM) v1

Understanding how in eukaryotic cells thousands of proteins are sorted from each other through the secretory pathway and delivered to their correct destinations is a central issue of cell biology. We have further investigated in yeast how two distinct types of cargo proteins are sorted into different endoplasmic reticulum (ER) exit sites (ERES) for their differential ER export to the Golgi apparatus. We used an optimized protocol that combines a live cell dual-cargo ER export system with a 3D simultaneous multi-color high-resolution live cell microscopy called Super-resolution Confocal Live Imaging Microscopy (SCLIM). Here, we describe this protocol, which is based on the reversible ER retention of two de novo co-expressed cargos by blocking COPII function upon incubation of the thermo-sensitive COPII allele sec31-1 at restrictive temperature (37ºC). ER export is restored by shifting down to permissive temperature (24ºC) and progressive incorporation of the two different types of cargos into the fluorescently labeled ERES can be then simultaneously captured at 3D high spatial resolution by SCLIM microscopy. By using this protocol, we have shown that newly synthesized glycosylphosphatidylinositol (GPI)-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized ERES that are distinct from those used by transmembrane secretory proteins. Furthermore, we showed that the chain length of the ceramide is critical for this sorting selectivity. Therefore, thanks to the presented method we could obtain the first direct in vivo evidence for lipid chain length-based protein cargo sorting into selective ERES.

The RNA helicase Ddx52 functions as a growth switch in juvenile zebrafish

Vertebrate animals usually display robust growth trajectories during juvenile stages, and reversible suspension of this growth momentum by a single genetic determinant has not been reported. Here, we report a single genetic factor that is essential for juvenile growth in zebrafish. Using a forward genetic screen, we recovered a temperature-sensitive allele, pan (after Peter Pan), that suspends whole-organism growth at juvenile stages. Remarkably, even after growth is halted for a full 8-week period, pan mutants are able to resume a robust growth trajectory after release from the restrictive temperature, eventually growing into fertile adults without apparent adverse phenotypes. Positional cloning and complementation assays revealed that pan encodes a probable ATP-Dependent RNA Helicase (DEAD-Box Helicase 52; ddx52) that maintains the level of 47S precursor ribosomal RNA. Furthermore, genetic silencing of ddx52 and pharmacological inhibition of bulk RNA transcription similarly suspend the growth of flies, zebrafish and mice. Our findings reveal evidence that safe, reversible pauses of juvenile growth can be mediated by targeting the activity of a single gene, and that its pausing mechanism has high evolutionary conservation.

The environmental stress response regulates ribosome content in cell cycle-arrested S. cerevisiae

Temperature sensitive cell division cycle (cdc-ts) cells are unable to progress through the cell cycle at the restrictive temperature due to mutations in genes essential to cell cycle progress. Cells harboring cdc-ts mutations increase in cell volume upon arrest but eventually stop growing. We found that this attenuation in growth was due to selective downregulation of ribosome concentration. We saw similar ribosome downregulation in cells arrested in the cell cycle through alpha factor addition, rapamycin addition, and entrance into stationary phase. In all cell cycle arrests studied, cells activated the Environmental Stress Response (ESR), a key transcriptional response to many stressors in S. cerevisiae. When we combined cell cycle arrest with hyperactivation of the Ras/PKA pathway, ESR activation was prevented, cells were unable to downregulate their ribosomes, and cell viability was decreased. Our work uncovers a key role for the environmental stress response in coupling cell cycle progression to biomass accumulation.

Multiple nutritional phenotypes of fission yeast mutants defective in genes encoding essential mitochondrial proteins

Mitochondria are essential for regulation of cellular respiration, energy production, small molecule metabolism, anti-oxidation and cell ageing, among other things. While the mitochondrial genome contains a small number of protein-coding genes, the great majority of mitochondrial proteins are encoded by chromosomal genes. In the fission yeast Schizosaccharomyces pombe , 770 proteins encoded by chromosomal genes are located in mitochondria. Of these, 195 proteins, many of which are implicated in translation and transport, are absolutely essential for viability. We isolated and characterized eight temperature-sensitive ( ts ) strains with mutations in essential mitochondrial proteins. Interestingly, they are also sensitive to limited nutrition (glucose and/or nitrogen), producing low-glucose-sensitive and ‘super-housekeeping' phenotypes. They fail to produce colonies under low-glucose conditions at the permissive temperature or lose cell viability under nitrogen starvation at the restrictive temperature. The majority of these ts mitochondrial mutations may cause defects of gene expression in the mitochondrial genome. mrp4 and mrp17 are defective in mitochondrial ribosomal proteins. ppr3 is defective in rRNA expression, and trz2 and vrs2 are defective in tRNA maturation. This study promises potentially large dividends because mitochondrial quiescent functions are vital for human brain and muscle, and also for longevity.

A genetic screen for temperature-sensitive morphogenesis-defectiveCaenorhabditis elegansmutants

Morphogenesis involves coordinated cell migrations and cell shape changes that generate tissues and organs, and organize the body plan. Cell adhesion and the cytoskeleton are important for executing morphogenesis, but their regulation remains poorly understood. As genes required for embryonic morphogenesis may have earlier roles in development, temperature-sensitive embryonic-lethal mutations are useful tools for investigating this process. From a collection of ∼200 such Caenorhabditis elegans mutants, we have identified 17 that have highly penetrant embryonic morphogenesis defects after upshifts from the permissive to the restrictive temperature, just prior to the cell shape changes that mediate elongation of the ovoid embryo into a vermiform larva. Using whole genome sequencing, we identified the causal mutations in seven affected genes. These include three genes that have roles in producing the extracellular matrix, which is known to affect the morphogenesis of epithelial tissues in multicellular organisms: the rib-1 and rib-2 genes encode glycosyltransferases, and the emb-9 gene encodes a collagen subunit. We also used live imaging to characterize epidermal cell shape dynamics in one mutant, or1219ts, and observed cell elongation defects during dorsal intercalation and ventral enclosure that may be responsible for the body elongation defects. These results indicate that our screen has identified factors that influence morphogenesis and provides a platform for advancing our understanding of this fundamental biological process.

Yeast Rgd3 is a phospho-regulated F-BAR-containing RhoGAP involved in the regulation of Rho3 distribution and cell morphology

Polarized growth requires the integration of polarity pathways with the delivery of exocytic vesicles for cell expansion and counterbalancing endocytic uptake. In budding yeast, the myosin-V Myo2 is aided by the kinesin-related protein Smy1 in carrying out the essential Sec4-dependent transport of secretory vesicles to sites of polarized growth. Over-expression suppressors of a conditional myo2 smy1 mutant identified a novel F-BAR-containing RhoGAP, Rgd3, that has activity primarily on Rho3, but also Cdc42. Internally tagged Rho3 is restricted to the plasma membrane in a gradient corresponding to cell polarity that is altered upon Rgd3 over-expression. Rgd3 itself is localized to dynamic polarized vesicles that, while distinct from constitutive secretory vesicles, are dependent on actin and Myo2 function. In vitro Rgd3 associates with liposomes in a PIP2-enhanced manner. Further, the Rgd3 C-terminal region contains several phosphorylatable residues within a reported SH3-binding motif. An unphosphorylated mimetic construct is active and highly polarized, while the phospho-mimetic form is not. Rgd3 is capable of activating Myo2, dependent on its phospho-state and Rgd3 overexpression rescues aberrant Rho3 localization and cell morphologies seen at the restrictive temperature in the myo2 smy1 mutant. We propose a model where Rgd3 functions to modulate and maintain Rho3 polarity during growth.

IS26 Family Members IS257 and IS1216 Also Form Cointegrates by Copy-In and Targeted Conservative Routes

ABSTRACT IS26 has been shown to form cointegrates both by a copy-in mechanism involving one insertion sequence (IS) and a target and by a targeted conservative mechanism involving two ISs. IS26 is the flagship of a group of 65 bacterial ISs in the recently redefined IS6/IS26 family. Here, whether other family members can also use two mechanisms was examined using members of the IS257/IS431 and IS1216 isoform groups, which are associated with antibiotic resistance genes in staphylococci and enterococci, respectively. Transposases Tnp257 and Tnp1216 have 39% and 47% amino acid identities, respectively, with Tnp26 and are 62% identical to one another. Using a novel transposition assay, pUC-based plasmids carrying these ISs integrated into the chromosome of a temperature-sensitive polA Escherichia coli strain grown at the restrictive temperature. In the cointegrates, the plasmid carrying IS257 was flanked by various 8-bp target site duplications, consistent with random target selection. However, in a mating-out assay, only the targeted conservative reaction was detectable at a low frequency in a recA-negative E. coli strain, indicating that IS257 is at least 100-fold less active than IS26. For IS1216, in mating-out assays, both copy-in and targeted conservative cointegrate formation were detectable at frequencies similar to those observed for IS26. Duplication of various 8-bp target sites was detected for the copy-in route. For both IS257 and IS1216, when both of the plasmids carried an IS, the targeted conservative route occurred at a significantly higher frequency than the copy-in route, and only cointegrates formed by the conservative route were detected. IMPORTANCE IS26 differs from other studied ISs in the reactions that it can undertake. The differences make IS26 uniquely suited to its key role in the recruitment and spread of antibiotic resistance genes in Gram-negative bacteria. However, whether other ISs in the IS6/IS26 family can perform the same reactions is not known. IS257/IS431 and IS1216 isoforms found associated with antibiotic resistance genes in the Gram-positive bacteria staphylococci, enterococci, streptococci, and clostridia are related to IS26. However, the way that they move had not been investigated, limiting interpretation of their role in resistance gene dissemination and in the formation of cointegrates and complex resistance regions in staphylococci and enterococci. Here, they are shown to share the broad catalytic capabilities of IS26, demonstrating that it is likely that all members of the redefined IS6/IS26 family of bacterial ISs likewise are able to use both the copy-in and conservative routes.

Adaptive regrowth in respiratory deficient strain of yeast Saccharomyces cerevisiae due to deletion of YKU70 gene

It is known that significant causes of malignant tumors are destabilization of the nuclear genome and mitochondrial dysfunction. Adaptive regrowth in yeast colonies (the appearance of cell subpopulations more adapted to unfavorable conditions under conditions of the death of the original culture) is used as a model of the initial stages of carcinogenesis. To study the features of the formation of adaptive regrowth, a reparationdefective and respiratory-deficient yeast strain of Saccharomyces cerevisiae was created. The thermosensitive mutation in the yku70 gene was used as an inducer of nuclear genome instability (at 37 оC it causes cell cycle arrest due to a reduction of the length of telomeric regions of chromosomes). Damage to the mitochondrial DNA of the ∆yku70 strain led to its respiratory deficiency (petite mutation). The isolated petite mutant ∆yku70 strain was cultured at optimal 28 оC and restrictive 37 оC temperatures, the state of the cell suspension was evaluated by light and fluorescence microscopy, to determine the viability of cells was used the analysis of microcolonies growth. Isolation of adaptive regrowth clones and analysis of their properties by the method of serial dilutions were conducted. To assess the genome stability of selected clones of adaptive regrowth, PCR analysis of the microsatellite sequences YOR267C, SC8132X, SCPTSY7 was conducted. When culturing the petite mutant of the strain ∆yku70 at a restrictive temperature of 37 оC for 7 days, the formation of viable subpopulations was detected, which can overcome the arrest of the cell cycle in the G2 / M phase. Further analysis of the isolated clones of adaptive regrowth showed that they differ in cell survival at restrictive temperature, resistance to UV radiation and the ability to form adaptive regrowth on colonies. In the analysis of microsatellite repeats in adaptive regrowth clones, no manifestations of instability of the studied sequences were detected.