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Single-molecule switching nanoscopy overcomes the diffraction limit of light by stochastically switching single fluorescent molecules on and off, and then localizing their positions individually. Recent advances in this technique have greatly accelerated the data acquisition speed and improved the temporal resolution of super-resolution imaging. However, it has not been quantified whether this speed increase comes at the cost of compromised image quality. The spatial and temporal resolution depends on many factors, among which laser intensity and camera speed are the two most critical parameters. Here we quantitatively compare the image quality achieved when imaging Alexa Fluor 647-immunolabeled microtubules over an extended range of laser intensities and camera speeds using three criteria - localization precision, density of localized molecules, and resolution of reconstructed images based on Fourier Ring Correlation. We found that, with optimized parameters, single-molecule switching nanoscopy at high speeds can achieve the same image quality as imaging at conventional speeds in a 5-25 times shorter time period. Furthermore, we measured the photoswitching kinetics of Alexa Fluor 647 from single-molecule experiments, and, based on this kinetic data, we developed algorithms to simulate single-molecule switching nanoscopy images. We used this software tool to demonstrate how laser intensity and camera speed affect the density of active fluorophores and influence the achievable resolution. Our study provides guidelines for choosing appropriate laser intensities for imaging Alexa Fluor 647 at different speeds and a quantification protocol for future evaluations of other probes and imaging parameters.

Newly developed scientific complementary metal-oxide semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition, enlarge the field of view and increase the effective quantum efficiency in single-molecule switching nanoscopy. However, sCMOS-intrinsic pixel-dependent readout noise substantially lowers the localization precision and introduces localization artifacts. We present algorithms that overcome these limitations and that provide unbiased, precise localization of single molecules at the theoretical limit. Using these in combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at rates of up to 32 reconstructed images per second in fixed and living cells.

An aqueous enzymatic process assisted by ultrasound extraction (AEP-UE) was applied to the extraction of oil from flaxseed (Linum usitatissimum L.). The highest oil recovery of 68.1% was obtained when ground flaxseed was incubated with 130 U/g of cellulase, pectinase, and hemicellulase for 12h, at 45°C and pH 5.0. The IC(50) values of oil obtained by AEP-UE and organic solvent extraction (OSE), as measured by DPPH scavenging activity essay, were 2.27 mg/mL and 3.31 mg/mL. The AEP-UE-derived oil had a 1.5% higher content of unsaturated fatty acids than the OSE-derived oil. AEP-UE is therefore a promising environmentally friendly method for large-scale preparation of flaxseed oil., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Life cycle thinking (LCT) is one of the philosophies that has recently appeared in the context of the sustainable development. Some of the already existing tools and methods, as well as some of the recently emerged ones, which seek to understand, interpret and design the life of a product, can be included into the scope of the LCT philosophy. That is the case of the material and energy flow analysis (MEFA), a tool derived from the industrial metabolism definition. This paper proposes a methodology combining MEFA with another technique derived from sustainable development which also fits the LCT philosophy, the BAT (best available techniques) analysis. This methodology, applied to an industrial process, seeks to identify the so-called improvable flows by MEFA, so that the appropriate candidate BAT can be selected by BAT analysis. Material and energy inputs, outputs and internal flows are quantified, and sustainable solutions are provided on the basis of industrial metabolism. The methodology has been applied to an exemplary roof tile manufacture plant for validation. 14 Improvable flows have been identified and 7 candidate BAT have been proposed aiming to reduce these flows. The proposed methodology provides a way to detect improvable material or energy flows in a process and selects the most sustainable options to enhance them. Solutions are proposed for the detected improvable flows, taking into account their effectiveness on improving such flows., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Cancer registries usually exclude nonmelanoma skin cancers (NMSC), despite the large population affected. Health maintenance organization (HMO) and health system administrative databases could be used as sampling frames for ascertaining NMSC. NMSC patients diagnosed between January 1, 1988, and December 31, 2007, from such defined US populations were identified by using 3 algorithms: NMSC International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes, NMSC treatment Current Procedural Terminology (CPT) codes, or both codes. A subset of charts was reviewed to verify NMSC diagnosis, including all records from HMO-enrollee members in 2007. Positive predictive values for NMSC ascertainment were calculated. Analyses of data from 1988-2007 ascertained 11,742 NMSC patients. A random sample of 965 cases was selected for chart review, and NMSCs were validated in 47.0% of ICD-9-CM-identified patients, 73.4% of CPT-identified patients, and 94.9% identified with both codes. All charts from HMO-health plan enrollees in 2007 were reviewed (n = 1,116). Cases of NMSC were confirmed in 96.5% of ICD-9-CM-identified patients, 98.3% of CPT-identified patients, and 98.7% identified with both codes. HMO administrative data can be used to ascertain NMSC with high positive predictive values with either ICD-9-CM or CPT code, but both codes may be necessary among non-HMO patient populations.

Complex partial seizures, commonly arising from temporal lobe epilepsy (TLE), are associated with neuronal loss and post-seizure impairment of consciousness. We tested the hypothesis that TLE subjects, in between seizures, are associated with a decreased level of consciousness that is manifested by an enhanced response to a general anesthetic. Two animal models of TLE--amygdala kindling and pilocarpine-induced status epilepticus (Pilo-SE)--were tested. Pilo-SE rats, but not amygdala-kindled rats, showed a prolonged loss of pain and righting responses after 20 and 40 mg/kg i.p. pentobarbital, 2% halothane, and 5 and 10 mg/kg i.v. propofol as compared to control saline-treated rats. Since the major pathology of Pilo-SE rats was cell loss in the piriform cortex (PC) and the entorhinal cortex (EC), we studied the anesthetic response after inactivation of the EC or PC by locally infusing GABAA receptor agonist muscimol. Muscimol inactivation of the PC or EC, as compared to saline infusion in the same rats, prolonged the duration of loss of righting reflex, typically without changing the duration of loss of tail-pinch response, after 20 mg/kg i.p. pentobarbital, 2% halothane and 5 mg/kg i.v. propofol. Muscimol infusion, as compared to saline infusion, in the PC or EC also tended to decrease 30-100 Hz gamma EEG in the frontal cortex. In conclusion, a TLE model that resulted in neuronal loss, Pilo-SE, enhanced the response to a general anesthetic that could partly be attributed to a loss of neurons in the EC and PC.

Purpose: To study the behavioral depression induced by a generalized limbic seizure and by an opioid. The hypothesis that an opioid fentanyl and an amygdala-evoked seizure induced behavioral depression through the nucleus accumbens was tested., Methods: The behavioral depression induced by an amygdala-kindled seizure was studied in fully kindled rats, with or without prior injection of fentanyl (0.05 mg/kg, s.c.). Local infusion of a nonspecific opioid antagonist, naloxone, or saline, was made bilaterally in the nucleus accumbens. The durations of loss of righting reflex (LORR), loss of tail-pinch response, and catalepsy were assessed., Results: Fentanyl induced an LORR following a generalized kindled seizure. The combination of fentanyl and a generalized seizure, as compared to fentanyl alone or a generalized seizure alone, resulted in a prolonged duration of LORR, catalepsy, and loss of tail pinch response. Infusion of naloxone as compared to saline in the nucleus accumbens reduced the duration of catalepsy and LORR induced by fentanyl, with or without a generalized seizure., Conclusions: Postictal depression that follows a generalized kindled seizure enhanced the behavioral effects of the opioid fentanyl. Network synaptic depression induced by the seizure acted synergistically with fentanyl to produce analgesia, catalepsy, and LORR, in part through the nucleus accumbens.

The study of phenomena related to the dispersion of pollutants usually takes advantage of the use of mathematical models based on the description of the different processes involved. This educational approach is especially important in air pollution dispersion, when the processes follow a non-linear behaviour so it is difficult to understand the relationships between inputs and outputs, and in a 3D context where it becomes hard to analyze alphanumeric results. In this work, three different software tools, as computer solvers for typical air pollution dispersion phenomena, are presented. Each software tool developed to be implemented on PCs, follows approaches that represent three generations of programming languages (Fortran 77, VisualBasic and Java), applied over three different environments: MS-DOS, MS-Windows and the world wide web. The software tools were tested by students of environmental engineering (undergraduate) and chemical engineering (postgraduate), in order to evaluate the ability of these software tools to improve both theoretical and practical knowledge of the air pollution dispersion problem, and the impact of the different environment in the learning process in terms of content, ease of use and visualization of results.

Nuclear transcription is repressed when eukaryotic cells enter mitosis. Mitotic repression of transcription of various cellular and viral gene promoters by RNA polymerase II can be reproduced in vitro either with extracts prepared from cells arrested at mitosis with the microtubule polymerization inhibitor nocodazole or with nuclear extracts prepared from asynchronous cells and the mitotic protein kinase cdc2/cyclin B. Purified cdc2/cyclin B kinase is also sufficient to inhibit transcription in reconstituted transcription reactions with biochemically purified and recombinant basal transcription factors and RNA polymerase II. The cyclin-dependent kinase inhibitor p21Waf1/Cip1/Sdi1 can reverse the effect of cdc2/cyclin B kinase, indicating that repression of transcription is due to protein phosphorylation. Transcription rescue and inhibition experiments with each of the basal factors and the polymerase suggest that multiple components of the transcription machinery are inactivated by cdc2/cyclin B kinase. For an activated promoter, targets of repression are TFIID and TFIIH, while for a basal promoter, TFIIH is the major target for mitotic inactivation of transcription. Protein labeling experiments indicate that the p62 and p36 subunits of TFIIH are in vitro substrates for mitotic phosphorylation. Using the carboxy-terminal domain of the large subunit of RNA polymerase II as a test substrate for phosphorylation, the TFIIH-associated kinase, cdk7/cyclin H, is inhibited concomitant with inhibition of transcription activity. Our results suggest that there exist multiple phosphorylation targets for the global shutdown of transcription at mitosis.

In the C4 dicotyledonous grain plant amaranth (Amaranthus hypochondriacus), a mitochondrial NAD-dependent malic enzyme (NAD-ME; EC 1.1.1.39) serves a specialized and essential role in photosynthetic carbon fixation. In this study we have examined specialized photosynthetic gene expression patterns for the NAD-ME [alpha] subunit. We show here that the [alpha] subunit gene is preferentially expressed in leaves and cotyledons (the most photosynthetically active tissues), and this expression is specific to the bundle-sheath cells of these tissues from the earliest stages of development. Synthesis of the [alpha] subunit polypeptide and accumulation of its corresponding mRNA are strongly light-dependent, but this regulation is also influenced by seedling development. In addition, light-dependent accumulation of the [alpha] subunit mRNA is regulated at transcriptional as well as posttranscriptional levels. Our findings demonstrate that the NAD-ME of amaranth has acquired numerous complex tissue-specific and light-mediated regulation patterns that define its specialized function as a key enzyme in the C4 photosynthetic pathway.

In some C4 plant species, a mitochondrial NAD-dependent malic enzyme (EC 1.1.1.39) (NAD-ME) catalyzes the decarboxylation of 4 carbon malate in the bundle sheath cells, releasing CO2 for the Calvin cycle of photosynthesis. In amaranth, a dicotyledonous NAD-ME-type C4 plant, the photosynthetic NAD-ME purified as two subunits of 65 and 60 kDa, designated alpha and beta, respectively. Antiserum raised against the alpha subunit reacted only with the 65-kDa protein in immunoblot analysis. Immunogold electron microscopy using the alpha subunit antiserum demonstrated that this protein was localized specifically to the mitochondrial matrix of bundle sheath cells. The complete nucleotide sequence of a 2300-base pair alpha subunit cDNA clone showed that this gene encodes a protein that contains all of the motifs required for a complete and functional malic enzyme. The alpha subunit has significant similarity along its entire length to other known NAD- and NADP-dependent malic enzymes from plants, animals, and bacteria. The findings presented here provide new insights about the C4 photosynthetic NAD-ME and its evolutionary relationship to other forms of malic enzyme present in eukaryotic and prokaryotic organisms.

The patterns of expression for genes encoding several C4 photosynthetic enzymes were examined in light-grown and dark-grown (etiolated) cotyledons of amaranth (Amaranthus hypochondriacus), a dicotyledonous C4 plant. The large subunit and small subunit of ribulose-1,5-bisphosphate carboxylase (RuBPCase), phosphoenolpyruvate carboxylase (PEPCase), and pyruvate orthophosphate dikinase (PPdK) were all present in the cotyledons by d 2 after planting when the seedlings first emerged from the seed coat. Kranz anatomy was apparent in light-grown cotyledons throughout development, and the overall patterns of C4 gene expression were similar to those recently described for developing amaranth leaves (J.L. Wang, D.F. Klessig, J.O. Berry [1992] Plant Cell 4: 173-184). RuBPCase mRNA and proteins were present in both bundle sheath and mesophyll cells in a C3-like pattern during early development and became progressively more localized to bundle sheath cells in the C4-type pattern as the cotyledons expanded over 2 to 7 d. PEPCase and PPdK polypeptides were localized to mesophyll cells throughout development, even though PEPCase transcripts were detected in both bundle sheath and mesophyll cells. Kranz anatomy also developed in cotyledons grown in complete darkness. In 7-d-old dark-grown cotyledons, RuBPCase, PPdK, and PEPCase were all localized to the appropriate cell types, although at somewhat lower levels than in light-grown cotyledons. These findings demonstrate that the leaves and postembryonic cotyledons of amaranth undergo common developmental programs of C4 gene expression during maturation. Furthermore, light is not required for the cell-type-specific expression of genes encoding RuBPCase and other photosynthetic enzymes in this dicotyledonous C4 plant.