Corynebacterium glutamicum survives arsenic stress with arsenate reductases coupled to two distinct redox mechanisms

Arsenate reductases (ArsCs) evolved independently as a defence mechanism against toxic arsenate. In the genome of Corynebacterium glutamicum, there are two arsenic resistance operons (ars1 and ars2) and four potential genes coding for arsenate reductases (Cg_ArsC1, Cg_ArsC2, Cg_ArsC1' and Cg_ArsC4). Using knockout mutants, in vitro reconstitution of redox pathways, arsenic measurements and enzyme kinetics, we show that a single organism has two different classes of arsenate reductases. Cg_ArsC1 and Cg_ArsC2 are single-cysteine monomeric enzymes coupled to the mycothiol/mycoredoxin redox pathway using a mycothiol transferase mechanism. In contrast, Cg_ArsC1' is a three-cysteine containing homodimer that uses a reduction mechanism linked to the thioredoxin pathway with a k(cat)/K(M) value which is 10(3) times higher than the one of Cg_ArsC1 or Cg_ArsC2. Cg_ArsC1' is constitutively expressed at low levels using its own promoter site. It reduces arsenate to arsenite that can then induce the expression of Cg_ArsC1 and Cg_ArsC2. We also solved the X-ray structures of Cg_ArsC1' and Cg_ArsC2. Both enzymes have a typical low-molecular-weight protein tyrosine phosphatases-I fold with a conserved oxyanion binding site. Moreover, Cg_ArsC1' is unique in bearing an N-terminal three-helical bundle that interacts with the active site of the other chain in the dimeric interface.     

Fluctuations at a Low Mean Temperature Accelerate Dengue Virus Transmission by Aedes aegypti

Background

Environmental factors such as temperature can alter mosquito vector competence for arboviruses. Results from recent studies indicate that daily fluctuations around an intermediate mean temperature (26°C) reduce vector competence of Aedes aeygpti for dengue viruses (DENV). Theoretical predictions suggest that the mean temperature in combination with the magnitude of the diurnal temperature range (DTR) mediate the direction of these effects.

Methodology/Principal Findings

We tested the effect of temperature fluctuations on Ae. aegypti vector competence for DENV serotype-1 at high and low mean temperatures, and confirmed this theoretical prediction. A small DTR had no effect on vector competence around a high (30°C) mean, but a large DTR at low temperature (20°C) increased the proportion of infected mosquitoes with a disseminated infection by 60% at 21 and 28 days post-exposure compared to a constant 20°C. This effect resulted from a marked shortening of DENV extrinsic incubation period (EIP) in its mosquito vector; i.e., a decrease from 29.6 to 18.9 days under the fluctuating vs. constant temperature treatment.

Conclusions

Our results indicate that Ae. aegypti exposed to large fluctuations at low temperatures have a significantly shorter virus EIP than under constant temperature conditions at the same mean, leading to a considerably greater potential for DENV transmission. These results emphasize the value of accounting for daily temperature variation in an effort to more accurately understand and predict the risk of mosquito-borne pathogen transmission, provide a mechanism for sustained DENV transmission in endemic areas during cooler times of the year, and indicate that DENV transmission could be more efficient in temperate regions than previously anticipated.     

Intranasal immunization with temperature-sensitive mutants protects granulocytopenic mice from lethal pulmonary challenge withPseudomonas aeruginosa

Intranasal (i.n.) immunization with two temperature-sensitive (ts) mutants ofPseudomonas aeruginosa protected, in a dose-related manner, granulocytopenic (GCP) mice challenged with a lethal dose of the wild-type (wt) organism. The number of ts mutants in oronasopharyngeal lavage fluids and stools decreased steadily in both normal and GCP mice after i.n. immunization. Intranasal immunization with 10⁷ colony-forming units (CFU) of either mutant induced significant protection, whereas intraperitoneal (i.p.) immunization with similar doses induced lower protection. Protection induced by i.n. immunization was accompanied by increased levels of anti-P. aeruginosa IgA in lung lavage fluids. The results of this study demonstrate the usefulness of ts mutants ofP. aeruginosa for local immunization to protect GCP hosts from fatalP. aeruginosa pneumonia.     

Interplay between cytochrome c and gibberellins during Arabidopsis vegetative development

We studied the effect of reducing the levels of the mitochondrial electron carrier cytochrome c (CYTc) in Arabidopsis thaliana. Plants with CYTc deficiency have delayed growth and development, and reach flowering several days later than the wild‐type but with the same number of leaves. CYTc‐deficient plants accumulate starch and glucose during the day, and contain lower levels of active gibberellins (GA) and higher levels of DELLA proteins, involved in GA signaling. GA treatment abolishes the developmental delay and reduces glucose accumulation in CYTc‐deficient plants, which also show a lower raise in ATP levels in response to glucose. Treatment of wild‐type plants with inhibitors of mitochondrial energy production limits plant growth and increases the levels of DELLA proteins, thus mimicking the effects of CYTc deficiency. In addition, an increase in the amount of CYTc decreases DELLA protein levels and expedites growth, and this depends on active GA synthesis. We conclude that CYTc levels impinge on the activity of the GA pathway, most likely through changes in mitochondrial energy production. In this way, hormone‐dependent growth would be coupled to the activity of components of the mitochondrial respiratory chain.     

Notch3 marks clonogenic mammary luminal progenitor cells in vivo

The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive “triple negative” human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2^SAT transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells.     

Olfactory neurons expressing identified receptor genes project to subsets of glomeruli within the antennal lobe of Drosophila melanogaster

We have used green fluorescent protein to trace the projection patterns of olfactory neurons expressing identified candidate odorant receptors to the brain of Drosophila. At the periphery, receptor expression correlates with specific sense-organ subtype, independent of location on the antennal surface. The majority of neurons expressing a given receptor converge onto one or two major glomeruli as described previously. However, we detected a few additional glomeruli, which are less intensely innervated and also tend to be somewhat variable. This means that functionally similar olfactory neurons connect to small subsets of glomeruli rather than to a single glomerulus as believed previously. This finding has important implications for our understanding of odor coding and the generation of olfactory behavior.     

A thermoplastic microfluidic microphysiological system to recapitulate hepatic function and multicellular interactions

Hepatic in vitro platforms ranging from multi-well cultures to bioreactors and microscale systems have been developed as tools to recapitulate cellular function and responses to aid in drug screening and disease model development. Recent developments in microfabrication techniques and cellular materials enabled fabrication of next-generation, advanced microphysiological systems (MPSs) that aim to capture the cellular complexity and dynamic nature of the organ presenting highly controlled extracellular cues to cells in a physiologically relevant context. Historically, MPSs have heavily relied on elastomeric materials in their manufacture, with unfavorable material characteristics (such as lack of structural rigidity) limiting their use in high-throughput systems. Herein, we aim to create a microfluidic bilayer model (microfluidic MPS) using thermoplastic materials to allow hepatic cell stabilization and culture, retaining hepatic functional phenotype and capturing cellular interactions. The microfluidic MPS consists of two overlapping microfluidic channels separated by a porous tissue-culture membrane that acts as a surface for cellular attachment and nutrient exchange; and an oxygen permeable material to stabilize and sustain primary human hepatocyte (PHH) culture. Within the microfluidic MPS, PHHs are cultured in the top channel in a collagen sandwich gel format with media exchange accomplished through the bottom channel. We demonstrate PHH culture for 7 days, exhibiting measures of hepatocyte stabilization, secretory and metabolic functions. In addition, the microfluidic MPS dimensions provide a reduced media-to-cell ratio in comparison with multi-well tissue culture systems, minimizing dilution and enabling capture of cellular interactions and responses in a hepatocyte-Kupffer coculture model under an inflammatory stimulus. Utilization of thermoplastic materials in the model and ability to incorporate multiple hepatic cells within the system is our initial step towards the development of a thermoplastic-based high-throughput microfluidic MPS platform for hepatic culture. We envision the platform to find utility in development and interrogation of disease models of the liver, multi-cellular interactions and therapeutic responses.     

Kinetic properties and metabolic contributions of yeast mitochondrial and cytosolic NADP+-specific isocitrate dehydrogenases

To compare kinetic properties of homologous isozymes of NADP+-specific isocitrate dehydrogenase, histidine-tagged forms of yeast mitochondrial (IDP1) and cytosolic (IDP2) enzymes were expressed and purified. The isozymes were found to share similar apparent affinities for cofactors. However, with respect to isocitrate, IDP1 had an apparent Km value approximately 7-fold lower than that of IDP2, whereas, with respect to alpha-ketoglutarate, IDP2 had an apparent Km value approximately 10-fold lower than that of IDP1. Similar Km values for substrates and cofactors in decarboxylation and carboxylation reactions were obtained for IDP2, suggesting a capacity for bidirectional catalysis in vivo. Concentrations of isocitrate and alpha-ketoglutarate measured in extracts from the parental strain were found to be similar with growth on different carbon sources. For mutant strains lacking IDP1, IDP2, and/or the mitochondrial NAD+-specific isocitrate dehydrogenase (IDH), metabolite measurements indicated that major cellular flux is through the IDH-catalyzed reaction in glucose-grown cells and through the IDP2-catalyzed reaction in cells grown with a nonfermentable carbon source (glycerol and lactate). A substantial cellular pool of alpha-ketoglutarate is attributed to IDH function during glucose growth, and to both IDP1 and IDH function during growth on glycerol/lactate. Complementation experiments using a strain lacking IDH demonstrated that overexpression of IDP1 partially compensated for the glutamate auxotrophy associated with loss of IDH. Collectively, these results suggest an ancillary role for IDP1 in cellular glutamate synthesis and a role for IDP2 in equilibrating and maintaining cellular levels of isocitrate and alpha-ketoglutarate.     

Postnatal Hyperoxia Exposure Differentially Affects Hepatocytes and Liver Haemopoietic Cells in Newborn Rats

Premature newborns are frequently exposed to hyperoxic conditions and experimental data indicate modulation of liver metabolism by hyperoxia in the first postnatal period. Conversely, nothing is known about possible modulation of growth factors and signaling molecules involved in other hyperoxic responses and no data are available about the effects of hyperoxia in postnatal liver haematopoiesis. The aim of the study was to analyse the effects of hyperoxia in the liver tissue (hepatocytes and haemopoietic cells) and to investigate possible changes in the expression of Vascular Endothelial Growth Factor (VEGF), Matrix Metalloproteinase 9 (MMP-9), Hypoxia-Inducible Factor-1α (HIF-1α), endothelial Nitric Oxide Synthase (eNOS), and Nuclear Factor-kB (NF-kB). Experimental design of the study involved exposure of newborn rats to room air (controls), 60% O₂ (moderate hyperoxia), or 95% O₂ (severe hyperoxia) for the first two postnatal weeks. Immunohistochemical and Western blot analyses were performed. Severe hyperoxia increased hepatocyte apoptosis and MMP-9 expression and decreased VEGF expression. Reduced content in reticular fibers was found in moderate and severe hyperoxia. Some other changes were specifically produced in hepatocytes by moderate hyperoxia, i.e., upregulation of HIF-1α and downregulation of eNOS and NF-kB. Postnatal severe hyperoxia exposure increased liver haemopoiesis and upregulated the expression of VEGF (both moderate and severe hyperoxia) and eNOS (severe hyperoxia) in haemopoietic cells. In conclusion, our study showed different effects of hyperoxia on hepatocytes and haemopoietic cells and differential involvement of the above factors. The involvement of VEGF and eNOS in the liver haemopoietic response to hyperoxia may be hypothesized.     

Antagonistic Role of CotG and CotH on Spore Germination and Coat Formation in Bacillus subtilis

Spore formers are bacteria able to survive harsh environmental conditions by differentiating a specialized, highly resistant spore. In Bacillus subtilis, the model system for spore formers, the recently discovered crust and the proteinaceous coat are the external layers that surround the spore and contribute to its survival. The coat is formed by about seventy different proteins assembled and organized into three layers by the action of a subset of regulatory proteins, referred to as morphogenetic factors. CotH is a morphogenetic factor needed for the development of spores able to germinate efficiently and involved in the assembly of nine outer coat proteins, including CotG. Here we report that CotG has negative effects on spore germination and on the assembly of at least three outer coat proteins. Such negative action is exerted only in mutants lacking CotH, thus suggesting an antagonistic effect of the two proteins, with CotH counteracting the negative role of CotG.