Subcycle quantum electrodynamics.

Squeezed states of electromagnetic radiation have quantum fluctuations below those of the vacuum field. They offer a unique resource for quantum information systems and precision metrology, including gravitational wave detectors, which require unprecedented sensitivity. Since the first experiments on this non-classical form of light, quantum analysis has been based on homodyning techniques and photon correlation measurements. These methods currently function in the visible to near-infrared and microwave spectral ranges. They require a well-defined carrier frequency, and photons contained in a quantum state need to be absorbed or amplified. Quantum non-demolition experiments may be performed to avoid the influence of a measurement in one quadrature, but this procedure comes at the expense of increased uncertainty in another quadrature. Here we generate mid-infrared time-locked patterns of squeezed vacuum noise. After propagation through free space, the quantum fluctuations of the electric field are studied in the time domain using electro-optic sampling with few-femtosecond laser pulses. We directly compare the local noise amplitude to that of bare (that is, unperturbed) vacuum. Our nonlinear approach operates off resonance and, unlike homodyning or photon correlation techniques, without absorption or amplification of the field that is investigated. We find subcycle intervals with noise levels that are substantially less than the amplitude of the vacuum field. As a consequence, there are enhanced fluctuations in adjacent time intervals, owing to Heisenberg's uncertainty principle, which indicate generation of highly correlated quantum radiation. Together with efforts in the far infrared, this work enables the study of elementary quantum dynamics of light and matter in an energy range at the boundary between vacuum and thermal background conditions.

Bioremoval of copper by filamentous fungi isolated from contaminated soils of Puchuncaví-Ventanas Central Chile.

Pollution represents a high risk to plants, animals, and human beings, causing an imbalance and affecting the environment. Soil is considered a universal sink, containing the highest load of environmental pollution. Puchuncaví-Ventanas sector, decreed as a saturated contamination zone in 1993, is considered one of the most affected areas by industrial pollution and belongs to one of the 5 sacrifice zones of Chile. The localities of Puchuncaví and Ventanas have heavy metal pollution levels that exceed up to 99% of the limits allowed by Canadian standards. The objective of this study was to characterize heavy metal tolerance and removal potential of filamentous fungi isolated from polluted soils for their use in decontamination systems and in situ soil improvement. Six fungal strains were selected based on their tolerance and a high capability to accumulate heavy metals, achieving copper bioaccumulation of 84% (Mortierella sp. strain LG01), 49% (Clonostachys sp. strain CQ23) and 48-77.5% (Trichoderma sp. strain LM01A). Trichoderma sp. strain LM01A was able to remove 41% of copper from contaminated soil under ex situ conditions. Some fungal strains belong to beneficial fungal genera, which are used as bioproducts in agriculture. The results of this study highlighted the use of Trichoderma sp. in soils contaminated, which may be of special interest in agriculture due to the large amounts of copper sulfate still applied as a pesticide in Chile and the world.

Antimicrobial efficacy of in vitro and ex vivo photodynamic therapy using porphyrins against Moraxella spp. isolated from bovine keratoconjunctivitis.

Infectious bovine keratoconjunctivitis (IBK) is an ocular disease affecting bovine herds worldwide, and it causes significant economic loss. The etiologic agent of IBK is considered to be Moraxella bovis, but M. ovis and M. bovoculi are frequently recovered of animals presenting clinical signs of IBK. The therapeutic measures available for its control have limited efficacy. Antimicrobial photodynamic therapy (aPDT) using porphyrins as photosensitizing molecules is an alternative method that can be used to reduce microbial growth. We evaluated the antibacterial activity of aPDT using two water-soluble tetra-cationic porphyrins (H2TMeP and ZnTMeP) against 22 clinical isolates and standard strains of Moraxella spp. in vitro and in an ex vivo model. For the in vitro assay, 4.0 µM of porphyrin was incubated with approximately 1.0 × 104 CFU/mL of each Moraxella sp. isolate and exposed to artificial light for 0, 2.5, 5, and 7.5 min. Next, 50 µL of this solution was plated and incubated for 24 h until CFU measurement. For the ex vivo assay, corneas excised from the eyeballs of slaughtered cattle were irrigated with Moraxella spp. culture, followed by the addition of zinc(II) porphyrin ZnTMeP (4.0 µM). The corneal samples were irradiated for 0, 7.5, and 30 min, followed by swab collection, plating, and CFU count. The results demonstrated the in vitro inactivation of the strains and clinical isolates of Moraxella spp. after 2.5 min of irradiation using ZnTMeP, reaching complete inactivation until 7.5 min. In the ex vivo experiment, the use of ZnTMeP resulted in the most significant reduction in bacterial concentration after 30 min of irradiation. These results encourage future in vivo experiments to investigate the role of metalloporphyrin ZnTMeP in the inactivation of Moraxella spp. isolates causing IBK.

10 W CEP-stable few-cycle source at 2 µm with 100 kHz repetition rate.

We developed a high repetition rate optical parametric chirped-pulse amplification (OPCPA) laser system based on fiber-laser-seeded Innoslab to generate few-cycle pulses around 2 µm with passively stable carrier-envelope phase (CEP) by difference frequency generation (DFG). Incorporating a piezo mirror before the DFG stage permits rapid CEP control. The OPCPA system is seeded by a stable supercontinuum generated in bulk material with the picosecond Innoslab pulses. Few-cycle pulses with durations of 17 fs and energies of over 100 µJ were produced in a single OPCPA stage. Three different nonlinear crystals: BBO, BiBO, and LNB were tested in the final parametric amplifier, and their average power related limitations are addressed.

Photothermal deflectometry enhanced by total internal reflection enables non-invasive glucose monitoring in human epidermis.

We present TIR-PTD spectroscopy, an IR-pump/VIS-probe method for the measurement of IR absorption spectra by means of photothermal deflectometry (PTD) enhanced by total internal reflection (TIR). It overcomes the limitations of IR spectroscopy for the study of opaque samples and allows molecular fingerprinting of IR-active liquids or solids. Another important advantage of the presented approach over traditional IR spectroscopy methods is the ability to obtain IR information by means of VIS detection, which is generally much cheaper and easier to handle than IR detection. By application of mid-IR TIR-PTD spectroscopy on human skin in vivo, we are demonstrating the correlation between epidermal- and blood-glucose levels on a type 1 diabetic patient.

Geographical variability of bacterial communities of cryoconite holes of Andean glaciers.

Cryoconite holes, ponds full of melting water with sediment on the bottom, are hotspots of biodiversity on glacier surfaces and host dynamic micro-ecosystems. They have been extensively investigated in different areas of the world (e.g., the Arctic, Antarctic, Alps, and Himalaya), but so far no study has described the bacterial communities of the glaciers in the Andes, the world's longest mountain range. In this study, we describe the bacterial communities of three small (< 2 km2) high-elevation (< 4200 m a.s.l.) glaciers of the Central Andes (Iver, East Iver and Morado glaciers) and two large (> 85 km2) glaciers of the Patagonian Andes (Exploradores and Perito Moreno glaciers) whose ablation tongues reach low altitude (< 300 m a.s.l.). Results show that the bacterial communities were generally similar to those observed in the cryoconite holes of other continents, but with few cyanobacteria (0.5% of sequences). The most abundant orders were Betaproteobacteriales, Cytophagales, Chitinophagales, Acetobacterales, Frankiales, Armatimonadales, Sphingobacteriales, Rhizobiales, Bacteroidales, Sphingomonadales, and Micrococcales. The bacterial communities differed between glaciers and both water pH and O2 concentration appeared to influence the bacterial community composition. This work thus provides the first description of the bacterial communities in cryoconite holes of South American glaciers.

Influence of microorganisms and leaching on simazine attenuation in an agricultural soil.

Simazine is an s-triazine herbicide world widely used for the control of broadleaf weeds. The influence of leaching and microorganisms on simazine attenuation in an agricultural soil long-term treated with this herbicide was studied. To elucidate the leaching potential of simazine in this soil, undisturbed soil columns amended with simazine were placed in a specially designed system and an artificial precipitation was simulated. To evaluate the simazine removal by soil microorganisms, three soil microcosm sets were established: i) control soil; ii) soil subjected to gamma irradiation (γ-soil) and iii) γ-soil inoculated with the simazine-degrading bacterium Pseudomonas sp. strain MHP41. The simazine-degrading microorganisms in soil were estimated using an indicator for respiration combined with MPN enumeration. The simazine removal in soil was monitored by GC-ECD and HPLC. In this agricultural soil the leaching of the applied simazine was negligible. The gamma irradiation decreased in more than one order of magnitude the cultivable heterotrophic bacteria and reduced the simazine-degrading microorganisms. Simazine was almost completely depleted (97%) in control soil by natural attenuation after 23 d, whereas in γ-soil only 70% of simazine was removed. The addition of the simazine-degrading strain MHP41 to γ-soil restored and upheld high stable simazine catabolic microorganisms as well as increased the simazine removal (87%). The results indicated that simazine is subjected to microbial degradation with negligible leaching in this agricultural soil and pointed out the crucial role of native microbiota in the herbicide removal.

Proteins containing the UBA domain are able to bind to multi-ubiquitin chains.

The UBA domain is a motif found in a variety of proteins, some of which are associated with the ubiquitin-proteasome system. We describe the isolation of a fission-yeast gene, mud1+, which encodes a UBA domain containing protein that is able to bind multi-ubiquitin chains. We show that the UBA domain is responsible for this activity. Two other proteins containing this motif, the fission-yeast homologues of Rad23 and Dsk2, are also shown to bind multi-ubiquitin chains via their UBA domains. These two proteins are implicated, along with the fission-yeast Pus1(S5a/Rpn10) subunit of the 26 S proteasome, in the recognition and turnover of substrates by this proteolytic complex.

Estrogen reduces neuronal generation of Alzheimer beta-amyloid peptides.

Alzheimer's disease (AD) is characterized by the accumulation of cerebral plaques composed of 40- and 42-amino acid beta-amyloid (Abeta) peptides, and autosomal dominant forms of AD appear to cause disease by promoting brain Abeta accumulation. Recent studies indicate that postmenopausal estrogen replacement therapy may prevent or delay the onset of AD. Here we present evidence that physiological levels of 17beta-estradiol reduce the generation of Abeta by neuroblastoma cells and by primary cultures of rat, mouse and human embryonic cerebrocortical neurons. These results suggest a mechanism by which estrogen replacement therapy can delay or prevent AD.

Isolation of Moraxella spp. from horses with conjunctivitis in Southern Brazil.

Infectious keratoconjunctivitis (IKC) is the most frequent ocular disease in livestock worldwide and is primarily caused by Moraxella bovis, M. ovis, and/or M. bovoculi. The economic impact of IKC is mainly due to ocular damage, which leads to weight loss, management difficulties, pain and discomfort, and cost of treatments. In horses, limited information is available on the association of Moraxella spp. with keratoconjunctivitis. The present report describes two cases of equine keratoconjunctivitis caused by members of the genus Moraxella. Both animals presented with lacrimation, conjunctivitis, photophobia, mucoid or purulent secretions, blepharitis, and conjunctival hyperemia. The diagnosis of IKC was based on the epidemiological and clinical findings; the etiological agent was identified through bacteriological (culture and biochemistry assays) and molecular testing (PCR and nucleotide sequencing). Our study reports the isolation of Moraxella bovoculi (SBP 88/19) and a putative new species/mutant of Moraxella (SBP 39/19) recovered from ocular secretions in horses. Thus, we suggest the inclusion of Moraxella spp. infection in the differential diagnosis of conjunctivitis in horses in Southern Brazil.

In vitro antimicrobial photodynamic therapy using tetra-cationic porphyrins against multidrug-resistant bacteria isolated from canine otitis.

Antimicrobial-resistant bacteria have been frequently isolated from canine otitis. Photodynamic therapy using porphyrins as photosensitizing molecules is an alternative therapy against microorganisms in localized infections. Therefore, we evaluated the antibacterial activity of two tetra-cationic porphyrins (H2TMeP and ZnTMeP) against gram-positive and -negative bacteria isolated from canine otitis, as well as its putative action mechanism. For this, two gram-positive and two gram-negative bacteria frequently detected in cases of canine otitis (coagulase-positive and -negative staphylococci [CPS and CNS], Pseudomonas aeruginosa, and Proteus mirabilis) were used in antibacterial activity assays. Each porphyrin at a non-cytotoxic dose was incubated with a fixed concentration of each bacterium and exposed to white-light irradiation for 0, 30, 60, and 90 min. Clinical isolates of CPS and CNS were photo-inactivated after 30 min of white-light exposure by both porphyrins (p < 0.05). Gram-negative bacteria were also photo-inactivated after 30 min (p < 0.05), reaching complete inactivation after 60 and 90 min of white-light irradiation by H2TMeP and ZnTMeP, respectively. Antibacterial assays using standard bacterial strains (ATCCs) demonstrated similar results to those obtained with clinical isolates, except for P. aeruginosa, which was completely inactivated by ZnTMeP at 60 min, and the absence of a significant reduction in P. mirabilis concentration when irradiated for 30 min. Similar assays were conducted using reactive oxygen species scavengers showing that the putative mechanism for bacterial inactivation is through the production of singlet oxygen species. These results indicated that H2TMeP and ZnTMeP tetra-cationic porphyrins were effective against bacteria isolated from canine otitis.

Selective and immediate effects of clathrin heavy chain mutations on Golgi membrane protein retention in Saccharomyces cerevisiae.

The role of clathrin in retention of Golgi membrane proteins has been investigated. Prior work showed that a precursor form of the peptide mating pheromone alpha-factor is secreted by Saccharomyces cerevisiae cells which lack the clathrin heavy chain gene (CHC1). This defect can be accounted for by the observation that the Golgi membrane protein Kex2p, which initiates maturation of alpha-factor precursor, is mislocalized to the cell surface of mutant cells. We have examined the localization of two additional Golgi membrane proteins, dipeptidyl aminopeptidase A (DPAP A) and guanosine diphosphatase (GDPase) in clathrin-deficient yeast strains. Our findings indicate that DPAP A is aberrantly transported to the cell surface but GDPase is not. In mutant cells carrying a temperature-sensitive allele of CHC1 (chc1-ts), alpha-factor precursor appears in the culture medium within 15 min, and Kex2p and DPAP A reach the cell surface within 30 min, after imposing the nonpermissive temperature. In contrast to these immediate effects, a growth defect is apparent only after 2 h at the nonpermissive temperature. Also, sorting of the vacuolar membrane protein, alkaline phosphatase, is not affected in chc1-ts cells until 2 h after the temperature shift. A temperature-sensitive mutation which blocks a late stage of the secretory pathway, sec1, prevents the appearance of mislocalized Kex2p at the cell surface of chc1-ts cells. We propose that clathrin plays a direct role in the retention of specific proteins in the yeast Golgi apparatus, thereby preventing their transport to the cell surface.

Clathrin-dependent localization of alpha 1,3 mannosyltransferase to the Golgi complex of Saccharomyces cerevisiae.

Posttranslational modification of yeast glycoproteins with alpha 1,3-linked mannose is initiated within a Golgi compartment analogous to the medial Golgi cisternae of higher eukaryotes. We have characterized the synthesis, posttranslational modification, and localization of the yeast alpha 1,3 mannosyltransferase (Mnn1p) using antibodies prepared against a segment of this protein expressed in bacteria. Mnn1p is initially synthesized as a 98.5-kD, type II integral membrane glycoprotein that is modified with both N- and O-linked oligosaccharides. It is subject to a slow, incremental increase in molecular mass that is dependent upon protein transport to the Golgi complex. Self-modification of Mnn1p with alpha 1,3 mannose epitopes, primarily on O-linked oligosaccharides, is at least partly responsible for the incremental increase in molecular mass. Mnn1p is a resident protein of the Golgi complex and colocalizes with guanosine diphosphatase to at least two physically distinct Golgi compartments by sucrose gradient fractionation, one of which may be a late Golgi compartment that also contains the Kex2 endopeptidase. Surprisingly, we found that a significant fraction of Mnn1p is mislocalized to the plasma membrane in a clathrin heavy chain temperature sensitive mutant while guanosine diphosphatase remains intracellular. A mutant Mnn1p that lacks the NH2-terminal cytoplasmic tail is properly localized to the Golgi complex, indicating that clathrin does not mediate Mnnlp Golgi retention by a direct interaction with the Mnn1p cytoplasmic tail. These results indicate that clathrin plays a broader role in the localization of Golgi proteins than anticipated.

Measured turbulent mixing in a small-scale circuit breaker model.

The performance of high voltage gas circuit breakers depends on the temperature distribution of hot gas or plasma from the arc zone mixed with cold gas that is present, for example, in the exhausts and mixing volume. Understanding the details of the mixing process is imperative to estimate the temperature distribution within the entire breaker volume. Design studies rely on computational fluid dynamics (CFD) simulations to search for the best way to achieve satisfactory mixing. One key uncertainty in the CFD simulations is the role of turbulence in this process and how to properly account for it. To gain knowledge of the mixing process between hot and cold gases, we have constructed a simplified breaker geometry that is flexible and accessible to diagnostics. Apart from standard measurements of current and arc voltage, we measure pressure in the arc zone and the mixing volume. Further, the mixing volume is specially designed to be transparent, allowing us to make shadowgraphy measurements of the turbulent mixing during and after the arcing phase. We report on experiments performed in air at atmospheric pressure.

Guidance cues at the Drosophila CNS midline: identification and characterization of two Drosophila Netrin/UNC-6 homologs.

Netrins are chemotropic guidance signals that play important roles in circumferential axon guidance in C. elegans and in the developing vertebrate spinal cord. We have identified two Drosophila homologs of this protein family (Netrin-A and Netrin-B). Both Netrins are dynamically expressed throughout embryogenesis, including CNS midline expression at the time of commissure formation. Both Netrin genes map close to each other on the X chromosome, and embryos deficient for this region exhibit defects in commissure formation. This CNS phenotype can be rescued by expression of either Netrin at the CNS midline, confirming an important role for Drosophila Netrins in commissural growth cone guidance. A localized source of Netrin protein at the midline is apparently important for function, since ectopic expression of either Netrin throughout the CNS results in phenotypic defects similar to the loss-of-function phenotype.

Mutations affecting growth cone guidance in Drosophila: genes necessary for guidance toward or away from the midline.

We performed a large-scale screen for mutations that affect the development of CNS axon pathways in the Drosophila embryo. We screened embryos from over 13,500 balanced lines and saved over 250 mutant lines whose phenotypes suggest possible defects in growth cone guidance. Here we focus on two new genes: commissureless (comm) and roundabout (robo). Mutations in comm lead to an absence of nearly all CNS axon commissures, such that growth cones that normally project across the midline instead now extend only on their own side. Mutations in robo lead to the opposite misrouting, such that some growth cones that normally extend only on their own side instead now project across the midline. The phenotypes of these two genes suggest that they may encode components of attractive and repulsive signaling systems at the midline that either guide growth cones across the midline or keep them on their own side.

commissureless controls growth cone guidance across the CNS midline in Drosophila and encodes a novel membrane protein.

The commissureless (comm) gene was identified previously in a large-scale screen for mutations that disrupt CNS axon pathways in Drosophila. The comm gene has a unique mutant phenotype: the complete absence of most axon commissures, while midline cells and other aspects of CNS fate and patterning are left unchanged. Here, we report on the molecular cloning, characterization, and expression of the comm gene. comm encodes a novel protein of 370 amino acids that lacks a signal sequence, has a transmembrane domain, and biochemically copurifies with membranes. COMM mRNA and COMM protein are dynamically expressed during embryogenesis, including by CNS midline glia during the formation of the axon commissures. Anti-COMM antibodies reveal strong staining of organelles likely to include the Golgi complex and endosomes and weaker staining of the cell surface. As commissural growth cones contact and traverse the CNS midline, COMM protein is apparently transferred from midline glia to commissural axons.

Dosage-sensitive, reciprocal genetic interactions between the Abl tyrosine kinase and the putative GEF trio reveal trio's role in axon pathfinding.

The Abelson tyrosine kinase (Abl) is integrated into signal transduction networks regulating axon outgrowth. We have identified the Drosophila trio gene through a mutation that exacerbates the Abl mutant phenotype. Drosophila Trio is an ortholog of mammalian Trio, a protein that contains multiple spectrin-like repeats and two Dbl homology (DH) domains that affect actin cytoskeletal dynamics via the small GTPases Rho and Rac. Phenotypic analysis demonstrates that trio and Abl cooperate in regulating axon outgrowth in the embryonic central nervous system (CNS). Dosage-sensitive interactions between trio and Abl, failed axon connections (fax), and enabled (ena) indicate that Trio is integrated into common signaling networks with these gene products. These observations suggest a mechanism by which Abl-mediated signaling networks influence the actin cytoskeleton in neuronal growth cones.

Protein topology of presenilin 1.

Mutations in a gene encoding a multitransmembrane protein, termed presenilin 1 (PS1), are causative in the majority of early-onset cases of AD. To determine the topology of PS1, we utilized two strategies: first, we tested whether putative transmembranes are sufficient to export a protease-sensitive substrate across a lipid bilayer; and second, we examined the binding of antibodies to specific PS1 epitopes in cultured cells selectively permeabilized with the pore-forming toxin, streptolysin-O. We document that the "loop," N-terminal, and C-terminal domains of PS1 are oriented toward the cytoplasm.