In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community.

Anaerobic bacterial biosynthesis of toluene from phenylacetate was reported more than two decades ago, but the biochemistry underlying this novel metabolism has never been elucidated. Here we report results of in vitro characterization studies of a novel phenylacetate decarboxylase from an anaerobic, sewage-derived enrichment culture that quantitatively produces toluene from phenylacetate; complementary metagenomic and metaproteomic analyses are also presented. Among the noteworthy findings is that this enzyme is not the well-characterized clostridial p-hydroxyphenylacetate decarboxylase (CsdBC). However, the toluene synthase under study appears to be able to catalyze both phenylacetate and p-hydroxyphenylacetate decarboxylation. Observations suggesting that phenylacetate and p-hydroxyphenylacetate decarboxylation in complex cell-free extracts were catalyzed by the same enzyme include the following: (i) the specific activity for both substrates was comparable in cell-free extracts, (ii) the two activities displayed identical behavior during chromatographic separation of cell-free extracts, (iii) both activities were irreversibly inactivated upon exposure to O2, and (iv) both activities were similarly inhibited by an amide analog of p-hydroxyphenylacetate. Based upon these and other data, we hypothesize that the toluene synthase reaction involves a glycyl radical decarboxylase. This first-time study of the phenylacetate decarboxylase reaction constitutes an important step in understanding and ultimately harnessing it for making bio-based toluene.

Ovarian function after hematopoietic cell transplantation: a descriptive study following the use of GnRH agonists for myeloablative conditioning and observation only for reduced-intensity conditioning.

Gonadal failure is a health and quality-of-life concern in hematopoietic cell transplant (HCT) survivors. While ovarian dysfunction is nearly universal following myeloablative (MA) conditioning, the risk is unclear after reduced-intensity conditioning (RIC). Gonadotropin-releasing hormone agonists decrease ovarian failure rates following conventional chemotherapy, but little is known about its effectiveness with HCT. We investigated the impact of leuprolide on ovarian function after MA conditioning and monitored ovarian function after RIC in this descriptive pilot study. Post-menarchal females <50 years undergoing HCT with adequate baseline ovarian function (follicle-stimulating hormone (FSH) level <40 mIU/mL and normal menstruation) were eligible. Prior to MA conditioning, leuprolide was administered. Those undergoing RIC were observed. FSH was measured at various time points. Seventeen women aged 12-45 years were evaluated (7 in the intervention group and 10 in the observation group). Compared to the historical high rate of ovarian failure after MA conditioning, 3 of 7 evaluable Lupron recipients had ovarian failure at a median of 703 days post transplant. Ovarian failure occurred in 1 of 10 recipients of RIC at a median follow-up of 901 days. In conclusion, leuprolide may protect ovarian function after MA conditioning. Additionally, RIC with cyclophosphamide, fludarabine and low-dose TBI has a low risk of ovarian failure.

Dense WDM transmission at 2 µm enabled by an arrayed waveguide grating.

We show, for the first time, dense WDM (8×20 Gbit/s) transmission at 2 µm enabled by advanced modulation formats (4-ASK Fast-OFDM) and the development of key components, including a new arrayed waveguide grating (AWGr) at 2 µm. The AWGr shows -12.8±1.78 dB of excess loss with an 18-dB extinction ratio and a thermal tunability of 0.108 nm/°C.

100 Gbit/s WDM transmission at 2 µm: transmission studies in both low-loss hollow core photonic bandgap fiber and solid core fiber.

We show for the first time 100 Gbit/s total capacity at 2 µm waveband, using 4 × 9.3 Gbit/s 4-ASK Fast-OFDM direct modulation and 4 × 15.7 Gbit/s NRZ-OOK external modulation, spanning a 36.3 nm wide wavelength range. WDM transmission was successfully demonstrated over 1.15 km of low-loss hollow core photonic bandgap fiber (HC-PBGF) and over 1 km of solid core fiber (SCF). We conclude that the OSNR penalty associated with the SCF is minimal, while a ~1-2 dB penalty was observed after the HC-PBGF probably due to mode coupling to higher-order modes.

Environmental modulation of microcystin and ß-N-methylamino-L-alanine as a function of nitrogen availability.

The most significant modulators of the cyanotoxins microcystin and ß-N-methylamino-L-alanine in laboratory cyanobacterial cultures are the concentration of growth-medium combined nitrogen and nitrogen uptake rate. The lack of field studies that support these observations led us to investigate the cellular content of these cyanotoxins in cyanobacterial bloom material isolated from a freshwater impoundment and to compare these to the combined nitrogen availability. We established that these toxins typically occur in an inverse relationship in nature and that their presence is mainly dependent on the environmental combined nitrogen concentration, with cellular microcystin present at exogenous combined nitrogen concentrations of 29 µM and higher and cellular BMAA correlating negatively with exogenous nitrogen at concentrations below 40 µM. Furthermore, opposing nutrient and light gradients that form in dense cyanobacterial blooms may result in both microcystin and BMAA being present at a single sampling site.

High-energy diode-seeded nanosecond 2 µm fiber MOPA systems incorporating active pulse shaping.

We report on high-energy nanosecond-pulsed fiber master oscillator power amplifier (MOPA) systems seeded by semiconductor laser diodes at 2 µm incorporating arbitrary pulse-shaping capabilities. Two MOPA systems, one based on direct diode modulation and the second using additional electro-optic modulator (EOM) based shaping, are investigated, with up to 0.5 mJ (25 kHz) and 1.0 mJ (12.5 kHz) pulse energies achieved, respectively, for 100 ns pulses with user-defined pulse shapes. Our results indicate that further energy scaling with shaped output pulses is primarily limited by the maximum pulse peak power available from the seed laser diode and the dynamic range offered by the first generation of EOMs at 2 µm.

100 kW peak power picosecond thulium-doped fiber amplifier system seeded by a gain-switched diode laser at 2 µm.

We report on the generation of picosecond pulses at 2 µm directly from a gain-switched discrete-mode diode laser and their amplification in a multistage thulium-doped fiber amplifier chain. The system is capable of operating at repetition rates in the range of 2 MHz-1.5 GHz without change of configuration, delivering high-quality 33 ps pulses with up to 3.5 µJ energy and 100 kW peak power, as well as up to 18 W of average power. These results represent a major technological advance and a 1 order of magnitude increase in peak power and pulse energy compared to existing picosecond sources at 2 µm.

Demonstration of amplified data transmission at 2 µm in a low-loss wide bandwidth hollow core photonic bandgap fiber.

The first demonstration of a hollow core photonic bandgap fiber (HC-PBGF) suitable for high-rate data transmission in the 2 µm waveband is presented. The fiber has a record low loss for this wavelength region (4.5 dB/km at 1980 nm) and a >150 nm wide surface-mode-free transmission window at the center of the bandgap. Detailed analysis of the optical modes and their propagation along the fiber, carried out using a time-of-flight technique in conjunction with spatially and spectrally resolved (S2) imaging, provides clear evidence that the HC-PBGF can be operated as quasi-single mode even though it supports up to four mode groups. Through the use of a custom built Thulium doped fiber amplifier with gain bandwidth closely matched to the fiber's low loss window, error-free 8 Gbit/s transmission in an optically amplified data channel at 2008 nm over 290 m of 19 cell HC-PBGF is reported.

Direct modulation of a tuneable slotted Fabry-Pérot laser with adaptive modulation OFDM.

Next generation optical access networks will require low cost lasers in conjunction with network flexibility and higher data rates. This work presents the direct modulation of a low cost tuneable slotted Fabry-Pérot laser (tuneable over 14 nm) with AM-OFDM. Characteristics of this dual section laser are presented and transmission of 10 Gb/s over 50 km is achieved with this device.

Application of serial sectioning FIB/SEM tomography in the comprehensive analysis of arrays of metal nanotubes.

The ever-increasing interest in nanostructured materials and shrinking dimensions of state-of-the-art devices pose new challenges both in synthesis and metrology. Although an extensive range of nanotubular materials of different compositions and for various applications are reported in the literature, often detailed structural characterisation of these materials is limited. This is due to the fact that techniques and characterisation protocols for structural analysis of 'buried' nano-scale features, defects or inhomogenities that are difficult to obtain by conventional imaging methods, are still not fully developed. In the case of 1D nanoporous structures, the continuity of the nano-tubular channels, their uniformity and orientation is of particular interest. Herein, we employ a serial sectioning technique on a dual beam FIB followed by 3D volume reconstruction for comprehensive analysis of tubular metal nanostructures encapsulated within porous anodic alumina. Using this technique, we demonstrate a nano-tomography characterisation protocol that can be used for analysis of nanoporous structures with emphasis on their channel uniformity and orientation. We demonstrate that high-resolution nano-tomography can be performed to visualise pores as small as 60 nm in diameter, with conical or globular shapes, and to quantitatively estimate their localisation and distribution along one-dimensional metal structures. We specifically chose to examine Cu-nanotubes, deposited electrochemically within anodic alumina template, because there is a great deal of debate regarding the deposition process. Hence, the comprehensive analysis shown here is not only demonstrating the applicability of the developed characterisation methodology but it is also, in conjunction with other advanced electron microscopy methods such as elemental nano-scale STEM/EDX mapping, providing conclusive evidence of the key factors at play during the deposition process.

Diversity and bioactive potential of endospore-forming bacteria cultured from the marine sponge Haliclona simulans.

AIMS: Despite the frequent isolation of endospore-formers from marine sponges, little is known about the diversity and characterization of individual isolates. The main aims of this study were to isolate and characterize the spore-forming bacteria from the marine sponge Haliclona simulans and to examine their potential as a source for bioactive compounds. METHODS AND RESULTS: A bank of presumptive aerobic spore-forming bacteria was isolated from the marine sponge H. simulans. These represented c. 1% of the total culturable bacterial population. A subgroup of thirty isolates was characterized using morphological, phenotypical and phylogenetic analysis. A large diversity of endospore-forming bacteria was present, with the thirty isolates being distributed through a variety of Bacillus and Paenibacillus species. These included ubiquitous species, such as B. subtilis, B. pumilus, B. licheniformis and B. cereus group, as well as species that are typically associated with marine habitats, such as B. aquimaris, B. algicola and B. hwajinpoensis. Two strains carried the aiiA gene that encodes a lactonase known to be able to disrupt quorum-sensing mechanisms, and various isolates demonstrated protease activity and antimicrobial activity against different pathogenic indicator strains, including Clostridium perfringens, Bacillus cereus and Listeria monocytogenes. CONCLUSIONS: The marine sponge H. simulans harbours a diverse collection of endospore-forming bacteria, which produce proteases and antibiotics. This diversity appears to be overlooked by culture-dependent and culture-independent methods that do not specifically target sporeformers. SIGNIFICANCE AND IMPACT OF STUDY: Marine sponges are an as yet largely untapped and poorly understood source of endospore-forming bacterial diversity with potential biotechnological, biopharmaceutical and probiotic applications. These results also indicate the importance of combining different methodologies for the comprehensive characterization of complex microbial populations such as those found in marine sponges.

Performance improvement of 10 Gb/s direct modulation OFDM by optical injection using monolithically integrated Discrete Mode lasers.

Performance improvement of a directly modulated 10 Gb/s OFDM system by optical injection of monolithically integrated lasers is shown experimentally over differing fibre lengths. The modulation and optical injection is performed using monolithically integrated Discrete Mode lasers. It is shown that optical injection with this device reduces third order inter-modulation distortion by up to 10dB and this results in an improvement in system performance from above a forward error correction BER threshold of 1 × 10(-3) to significantly below it.

Implementation of a cost-effective optical comb source in a WDM-PON with 10.7 Gb/s data to each ONU and 50 km reach.

The performance of a cost-effective optical comb source using commercial off the shelf (COTS) components in a WDM passive optical network is demonstrated. Eight comb modes are individually modulated at 10.7 Gb/s and transmitted over 50 km of single mode fiber for downlink transmission. Error free performance is obtained for each comb line and a maximum performance difference of 1.4 dB is experienced between the eight channels. Colorless operation of the optical network unit is achieved by utilizing an integrated module consisting of a tunable laser and an electro-absorption modulator as an uplink transmitter. Finally the predicted downstream performance of the system, when all the channels are transmitted simultaneously, is numerically simulated.

Exclusion of mouse hepatitis virus from a filtered, plastic rodent shipping container during an in transit field challenge.

Although leading suppliers of laboratory mice and rats continue to use filtered shipping boxes to protect their animals from contamination during transport to the end user, no information had been available in the literature to demonstrate that any of these boxes actually accomplish this task. To test this hypothesis, 12 plastic shipping boxes with filters and tight-fitting lids and six cardboard shipping boxes without filters (controls) were each stocked with adult, adventitious disease-free mice. All 18 shipping boxes were transported to a facility housing a breeding colony of mice enzootically infected with four murine viruses, including mouse hepatitis virus (MHV), and were placed inside the colony for 15 h. The boxes were then transported to a commercial testing laboratory, at which the animals were aseptically removed and were held in microisolation cages for 28 days, after which their sera tested for antibody to all four murine viruses. All serum samples from mice held in the control boxes were positive for antibody to MHV, whereas sera from all mice held in filtered boxes were negative for antibody to any of the four viruses. This study demonstrates that at least one type of filtered shipping container protects mice from a field challenge of MHV. To the best of our knowledge, this is the first documentation of any microbial efficacy testing conducted on filtered shipping containers for laboratory animals.

Antagonism of inhalant and volatile anesthetic enhancement of glycine receptor function.

Recent studies suggest that alcohols, volatile anesthetics, and inhaled drugs of abuse, which enhance gamma-aminobutyric acid, type A, and glycine receptor-activated ion channel function, may share common or overlapping molecular sites of action on these receptors. To investigate this possibility, these compounds were applied singly and in combination to wild-type glycine alpha(1) receptors expressed in Xenopus laevis oocytes. Data obtained from concentration-response curves of the volatile anesthetic enflurane constructed in the presence and absence of ethanol, chloroform, or toluene were consistent with competition for a common binding pocket on these receptors. A mutant glycine receptor, insensitive to the enhancing effects of ethanol but not anesthetics or inhalants, demonstrated antagonism of anesthetic and inhalant effects on this receptor. Although ethanol (25-200 mm) had no effect on its own in this receptor, it was able to inhibit reversibly the enhancing effect of enflurane, toluene, and chloroform in a concentration-dependent manner. These data suggest the existence of overlapping molecular sites of action for ethanol, inhalants, and volatile anesthetics on glycine receptors and illustrate the feasibility of pharmacological antagonism of the effects of volatile anesthetics.

Intra-VTA baclofen attenuates cocaine self-administration on a progressive ratio schedule of reinforcement.

The effect of microinjections of baclofen into the ventral tegmental area (VTA), nucleus accumbens, or striatum on cocaine self-administration reinforced on a progressive ratio schedule was investigated. Intra-VTA baclofen produced the most potent modulation of cocaine intake, causing a significant reduction in cocaine-reinforced break points at a dose (56 ng/side) that was substantially lower than doses necessary to produce comparable reductions in the nucleus accumbens or striatum. During the time that cocaine-reinforced responding was suppressed, rats demonstrated the capacity to complete several hundred responses on a concurrently available food-reinforced lever, indicating that the effect of baclofen on cocaine intake was not due to a generalized disruption in operant responding. The results indicate that GABA(B) receptors in the VTA may have the potential to modulate reward processes associated with cocaine use.

Effect of baclofen on cocaine self-administration in rats reinforced under fixed-ratio 1 and progressive-ratio schedules.

RATIONALE: Recent reports have indicated that the gamma-aminobutyric acid (GABA)B agonist baclofen attenuates the reinforcing effects of cocaine. OBJECTIVES: To further evaluate the effect of baclofen on cocaine self-administration under a fixed ratio (FR) and progressive ratio (PR) schedule of reinforcement. METHODS: In the first series of experiments, three dose-response curves were generated that examined the effect of three doses of baclofen (1.8, 3.2, or 5.6 mg/kg, i.p.) against four unit-injection doses of cocaine (0.19, 0.38, 0.75, and 1.5 mg/kg per injection) reinforced under a FRI schedule. For comparison, an additional group of rats was pretreated with haloperidol (32, 56, or 100 microg/kg, i.p.). A separate experiment examined the effect of baclofen (1.8, 3.2, or 5.6 mg/kg, i.p.) on responding for concurrently available cocaine or food reinforcement. RESULTS: Under the FR1 schedule, baclofen suppressed intake of low but not high unit injection doses of cocaine. In contrast to haloperidol, baclofen had no effect on the distribution of inter-injection intervals and, instead, produced long pauses in cocaine self-administration. Baclofen dose dependently reduced cocaine-reinforced responding on a PR schedule; concurrent access to a food-reinforced lever demonstrated that the animals retained the capacity to respond at high rates. CONCLUSION: The effect of baclofen pretreatment on cocaine self-administration is dependent on the unit injection dose of cocaine and on the response requirements of the schedule.

The GABA(B) agonist CGP 44532 decreases cocaine self-administration in rats: demonstration using a progressive ratio and a discrete trials procedure.

Previous self-administration experiments have shown that baclofen, the prototypical GABA(B) agonist, produces an apparent attenuation in the reinforcing effects of cocaine in rats. The present experiments examined the effects of CPG 44532, a novel and highly specific GABA(B) agonist, on cocaine self-administration using two distinctly different procedures. CGP 44532 (0.063-0.5 mg/kg) produced a dose dependent decrease in break point on a progressive-ratio (PR) schedule. A low dose of CGP 44532 (0.125 mg/kg) produced an apparent shift of the cocaine dose-response curve to the right. In contrast there was comparatively little effect on food-reinforced responding on the same PR schedule. Using a discrete-trials procedure that engendered a circadian pattern of self-administration, CPG 44532 (0.063-0.5 mg/kg) produced a dose-dependent suppression of cocaine intake in the 4 h period following treatment. When a concurrently available food reinforced lever was added to the discrete trials paradigm CGP 44532 failed to disrupt responding for food at any of the doses tested. Data from the PR and discrete-trials procedures taken together indicate that CGP 44532 produced a specific decrease in the motivation to self-administer cocaine.

Self-administration of cocaine analogs by rats.

RATIONALE: A novel scheme for the synthesis of cocaine analogs from vinylcarbenoid precursors has made available compounds that have a diverse range of affinities for the DA and 5-HT transporters. These compounds were used to explore the relationship between their biochemical properties and their reinforcing effects. OBJECTIVES: The objective was to assess the reinforcing efficacy of selected cocaine analogs and compare the results with their selectivity in binding to DA and 5-HT transporters. METHODS: Rats were prepared with chronically indwelling intravenous cannulae and trained to self-administer cocaine on a progressive ratio (PR) schedule. A range of doses of seven cocaine analogs were substituted for cocaine in separate groups of animals. RESULTS: The results demonstrate a wide range of reinforcing efficacies and potencies among the seven selected drugs. Four tropane analogs (WF-11, WF-23, WF-24, WF-55) were found to support self-administration behavior on a PR schedule while three did not (WF-31, WF-54 and WF-60). The DA/5-HT selectivity ratio was found to be a better predictor of self-administration behavior than affinity at the DA transporter alone. CONCLUSION: These data suggest that drugs with a higher affinity for the DA versus the 5-HT transporter are more likely to be self-administered.