The resolution dependence of optimal exposures in liquid nitrogen temperature electron cryomicroscopy of catalase crystals

Electron beam damage is the fundamental limit to resolution in electron cryomicroscopy (cryo-EM) of frozen, hydrated specimens. Radiation damage increases with the number of electrons used to obtain an image and affects information at higher spatial frequencies before low-resolution information. For the experimentalist, a balance exists between electron exposures sufficient to obtain a useful signal-to-noise ratio (SNR) in images and exposures that limit the damage to structural features. In single particle cryo-EM this balance is particularly delicate: low-resolution features must be imaged with a sufficient SNR to allow image alignment so that high-resolution features recorded below the noise level can be recovered by averaging independent images. By measuring the fading of Fourier components from images obtained at 200 kV of thin crystals of catalase embedded in ice, we have determined the electron exposures that will maximize the SNR at resolutions between 86 and 2.9 Å. These data allow for a rational choice of exposure for single particle cryo-EM. For example, for 20 Å resolution, the SNR is maximized at ～20 e^?/Ų, whereas for 3 Å resolution, it is maximized at ～10 e^?/Ų. We illustrate the effects of exposure in single particle cryo-EM with data collected at ～12–15 and ～24–30 e^?/Ų.     

Fabrication of carbon films with ∼500 nm holes for cryo-EM with a direct detector device

Single particle electron cryomicroscopy (cryo-EM) is often performed using EM grids coated with a perforated or holey layer of amorphous carbon. Regular arrays of holes enable efficient cryo-EM data collection and several methods for the production of micropatterned holey-carbon film coated grids have been described. However, a new generation of direct detector device (DDD) electron microscope cameras can benefit from hole diameters that are smaller than currently available. Here we extend a previously proposed method involving soft lithography with a poly(dimethylsiloxane) (PDMS) stamp for the production of holey-carbon film coated EM grids. By incorporating electron-beam (e-beam) lithography and modifying the procedure, we are able to produce low-cost high-quality holey-carbon film coated EM grids with ∼500 nm holes spaced 4 μm apart centre-to-centre. We demonstrate that these grids can be used for cryo-EM. Furthermore, we show that by applying image shifts to obtain movies of the carbon regions beside the holes after imaging the holes, the contrast transfer function (CTF) parameters needed for calculation of high-resolution cryo-EM maps with a DDD can be obtained efficiently.     

Radiation damage effects at four specimen temperatures from 4 to 100 K

Radiation damage is the primary factor that limits resolution in electron cryo-microscopy (cryo-EM) of frozen-hydrated biological samples. Negative effects of radiation damage are attenuated by cooling specimens to cryogenic temperatures using liquid nitrogen or liquid helium. We have examined the relationship between specimen temperature and radiation damage across a broad spectrum of resolution by analyzing images of frozen-hydrated catalase crystal at four specimen temperatures: 4, 25, 42, and 100 K. For each temperature, “exposure series” were collected consisting of consecutive images of the same area of sample, each with 10 e^?/Ų exposure per image. Radiation damage effects were evaluated by examining the correlation between cumulative exposure and normalized amplitudes or IQ values of Bragg peaks across a broad range of resolution (4.0–173.5 Å). Results indicate that for sub-nanometer resolution, liquid nitrogen specimen temperature (100 K) provides the most consistent high-quality data while yielding statistically equivalent protection from radiation damage compared to the three lower temperatures. At lower resolution, suitable for tomography, intermediate temperatures (25 or 42 K) may provide a modest improvement in cryo-protection without introducing deleterious effects evident at 4 K.     

Practical performance evaluation of a 10k × 10k CCD for electron cryo-microscopy

Electron cryo-microscopy (cryo-EM) images are commonly collected using either charge-coupled devices (CCD) or photographic film. Both film and the current generation of 16 megapixel (4k × 4k) CCD cameras have yielded high-resolution structures. Yet, despite the many advantages of CCD cameras, more than two times as many structures of biological macromolecules have been published in recent years using photographic film. The continued preference to film, especially for subnanometer-resolution structures, may be partially influenced by the finer sampling and larger effective specimen imaging area offered by film. Large format digital cameras may finally allow them to overtake film as the preferred detector for cryo-EM. We have evaluated a 111-megapixel (10k × 10k) CCD camera with a 9 μm pixel size. The spectral signal-to-noise ratios of low dose images of carbon film indicate that this detector is capable of providing signal up to at least 2/5 Nyquist frequency potentially retrievable for 3D reconstructions of biological specimens, resulting in more than double the effective specimen imaging area of existing 4k × 4k CCD cameras. We verified our estimates using frozen-hydrated ε15 bacteriophage as a biological test specimen with previously determined structure, yielding a ～7 ? resolution single particle reconstruction from only 80 CCD frames. Finally, we explored the limits of current CCD technology by comparing the performance of this detector to various CCD cameras used for recording data yielding subnanometer resolution cryo-EM structures submitted to the electron microscopy data bank (http://www.emdatabank.org/).     

Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film

Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry.     

Construction of a bacterial biosensor for zinc and copper and its application to the development of multifunctional heavy metal adsorption bacteria

In this study, we designed and applied molecular biosensors for heavy metals, zinc and copper, for use in bioremediation strategies. Bacteria utilize two component systems to sense changes in the environment by multiple signal components including heavy metals and control gene expression in response to changes in signal molecules. zraP and cusC promoters were selected from a genetic circuit of the ZraSR and CusSR two-component system and were fused to a dual-labeling reporter protein as an interactive biological component for zinc and copper to generate a signal from the constructed biosensor. The biosensor efficiently senses zinc and copper with a calculated detection limit of 16 μM and 26 μM, respectively, and was shown to be a sensitive and effective heavy metal monitoring bacterial system. To extend the application of the bacterial biosensor, we assembled a bioadsorption system that can trigger bacteria to sense and adsorb 13 ± 0.3 mg/L of zinc and 11.4 ± 0.42 mg/L of copper per gram of dry cell weight with induction at a concentration of 100 mg/L of the respective metal ion.     

Cryo-electron microscopy for structural analysis of dynamic biological macromolecules

Background

Since the introduction of what became today's standard for cryo-embedding of biological macromolecules at native conditions more than 30 years ago, techniques and equipment have been drastically improved and the structure of biomolecules can now be studied at near atomic resolution by cryo-electron microscopy (cryo-EM) while capturing multiple dynamic states. Here we review the recent progress in cryo-EM for structural studies of dynamic biological macromolecules.

Ethanol-to-methane activity of Geobacter-deprived anaerobic granules enhanced by conductive microparticles

Direct interspecies electron transfer (DIET) has been typically proposed as mechanism of electron transfer among methanogenic populations in granules during anaerobic digestion where Geobacter species play a key role. Using anaerobic granules where Geobacteraceae members were not prevalent − representing only 0.3% of total bacteria −, tests incubated with two co-substrates showed that the rate of methanogenesis from formate and hydrogen diminished in the presence of a non-methanogenic co-substrate such as ethanol. This could indicate that biological DIET occurs and competes with hydrogen and formate during methanogenesis. Moreover, the addition of conductive microparticles, such as stainless steel and granular activated carbon, was found to increase methanogenic activity in disintegrated granules by 190 ± 18% and 175 ± 22% respectively as compared to disintegrated granules devoid of microparticles. The addition of non-conductive microparticles such as porcelain however decreased methanogenic activity by 65 ± 3% of the disrupted granules without microparticle activity. These results indicate that syntrophic bacteria from anaerobic sludge excluding Geobacter species can also carry out conductive mineral mediated DIET.     

Initial evaluation of a direct detection device detector for single particle cryo-electron microscopy

We report on initial results of using a new direct detection device (DDD) for single particle reconstruction of vitreous ice embedded specimens. Images were acquired on a Tecnai F20 at 200 keV and a nominal magnification of 29,000×. This camera has a significantly improved signal to noise ratio and modulation transfer function (MTF) at 200 keV compared to a standard CCD camera installed on the same microscope. Control of the DDD has been integrated into Leginon, an automated data collection system. Using GroEL as a test specimen, we obtained images of ∼30 K particles with the CCD and the DDD from the same specimen sample using essentially identical imaging conditions. Comparison of the maps reconstructed from the CCD images and the DDD images demonstrates the improved performance of the DDD. We also obtained a 3D reconstruction from ∼70 K GroEL particles acquired using the DDD; the quality of the density map demonstrates the potential of this new recording device for cryoEM data acquisition.     

A highly sensitive and robust UPLC–MS with electrospray ionization method for quantitation of taxifolin in rat plasma

A sensitive ultra performance liquid chromatography–mass spectrometry method has been developed and validated for the quantification of taxifolin in rat plasma. Following liquid/liquid extraction by ethyl acetate, the analytes were separated on a Sunfire? (2.1 mm × 50 mm, 3.5 μm) column and analyzed in the selected ion recording with a negative electrospray ionization mode. The method was linear over the concentration range of 6–6750 ng/mL. Intra- and inter-day precisions were all within 8% and accuracy ranged from 92.9% to 105.1%. The lower limit of quantification was 6 ng/mL. The present method was successfully applied to the estimation of the pharmacokinetic parameters of taxifolin following intravenous and oral administration to rats. The absolute bioavailability of taxifolin was 0.17% in rat.     

Reaching the information limit in cryo-EM of biological macromolecules: experimental aspects

Although cryo-electron microscopy (cryo-EM) of biological macromolecules has made important advances in the past few years, the level of current technical performance is still well below what the physics of electron scattering would allow. It should be possible, for example, to use cryo-EM to solve protein structures at atomic resolution for particle sizes well below 80 kDa, but currently this has been achieved only for particles at least 10 times larger than that. In this review, we first examine some of the reasons for this large gap in performance. We then give an overview of work that is currently in progress to 1), improve the signal/noise ratio for area detectors; 2), improve the signal transfer between the scattered electrons and the corresponding images; and 3), reduce the extent to which beam-induced movement causes a steep fall-off of signal at high resolution. In each case, there is substantial reason to think that cryo-EM can indeed be made to approach the estimated physical limits.     

Acoustic patterns at the Samford Ecological Research Facility in South East Queensland,Australia: The Peri-Urban SuperSite of the Terrestrial Ecosystem Research Network

Acoustic signals that emanate from ecosystems are an important ecological variable which can provide evidence of current ecological condition as well as ecological change over time. The Terrestrial Ecosystem Research Network (TERN) established protocols to record sounds in ten SuperSites distributed throughout Australia with the objective of characterizing the soundscape in a representative landscape in different regions of Australia. This acoustic monitoring system enables a comparison of the soundscapes within and between Australian regions to determine similarities and differences in these landscapes and regions.This research quantifies the soundscape patterns in one of these SuperSites, Samford Ecological Research Facility (TERN-SERF), which is part of the South-East Queensland Peri-Urban SuperSite. An analysis and visualization of patterns in the soundscape was conducted using a continuous acoustic recording collected at TERN-SERF. The recording was made using a Song Meter (SM2) in a representative wooded habitat at TERN-SERF from 1 August to 30 September 2013. The recording was made in 16-bit stereo at 44 kHz and stored in wav file format. The recording was split into 1-minute-long recordings comprising 86,196 records and then sub-sampled at a 30-minute interval, providing 2878 one-minute-long recordings every 1/2 h. Soundscape metrics were computed for each of the two recording intervals. Soundscape power values were computed for each of ten frequency intervals (1–11 kHz) for both the 1-minute and the 30-minute interval recordings. In addition, six acoustic indices were computed from each recording.The acoustics metrics derived from the two sets of recordings (1-minute and 30-minute recording intervals) were examined to determine if they revealed different patterns. Several soundscape metrics were calculated for each recording including ten soundscape power values at 1 kHz frequency intervals and six acoustics indices. The soundscape shows a dynamic but consistent pattern over time of day during the monitoring period, depending on the metric examined. The metrics revealed different soundscape patterns. All soundscape power values at 1 kHz frequency intervals defined the dawn and dusk chorus, some more distinctly than others. Three of six acoustic indices also changed abruptly at the dawn chorus. No significant difference was found when soundscape metrics were compared between the 1-minute (high resolution) and 30-minute (lower resolution) recording intervals. A t-test was used to compare the mean values of ten soundscape power frequency intervals (p = 0.44) and the mean values of six acoustics indices (p = 0.41).Sounds were identified in 180 recordings made at 0530 h, 0600 h and 0630 h in the 1-minute long 30-minute interval recordings each day during the recording period (August and September). Sixty-seven species of birds were identified. Soundscape metrics were correlated with avian species counts and calls by all species using a correlation threshold of r > 0.7. This analysis revealed that soundscape power at the frequency interval 3–4 kHz was correlated with both the number of species (r = − 0.927) and total calls (r = − 0.996) over the three time periods. Three indices, the ADI (r = 0.953, r = 0.709), the AEI (r = 0.978, r = 0.774) and (H) (r = 0.795, r = 0.985) were similarly correlated as was an index derived soundscape power, the Shannon-Weaver Index (r = − 0.997, r = − 0.849). Other indices were correlated (r > 0.7) with only the number of avian species or only the number of calls.This methodology establishes an analysis protocol for analyzing large acoustic data sets, and demonstrates the effectiveness of using acoustic metrics for summarizing and interpreting long-term recordings.     

Soft X-ray tomography: virtual sculptures from cell cultures

(a) Cells are taken from their culture media, mounted on grids or in capillaries, and cryopreserved. Tomographic data are collected in a soft X-ray microscope with the cells maintained at cryogenic temperature. (b) A single slice through a 3D tomographic reconstruction of an HT1080 fibrosarcoma cell. The slice comprises tomographic reconstructions from two adjacent fields of view computationally stitched together. (c) 3D volume rendering of the tomographic reconstruction shown in (b) after segmentation guided by linear absorption coefficients (LAC) and organelle morphology. Key: Red; Nucleus, Turquoise; Mitochondria, Blue; Lipids, Gray; cell membrane. Scale bar = 1.5 μm.

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Development of Novel Glucose oxidase Immobilization on Graphene/Gold nanoparticles/Poly Neutral red modified electrode

Glucose oxidase (GOx) was immobilized onto glassy carbon electrode (GCE) that modified by reduced graphene oxide-gold nanoparticles- poly neutral red (RGO/AuNPs/PNR) nanocomposite. The composite was analyzed by scanning electron microscope (SEM), energy dispersive x-ray (EDX) spectroscopy, atomic force microscopy (AFM), attenuated total reflectance (ATR), cyclic voltammetry (CV), chronoamperometry and electrochemical impedance spectroscopy (EIS). SEM/EDX analysis showed the morphological of the nanocomposite. AFM results showed the morphology and structure of the RGO/AuNPs and RGO surfaces. The covalent bonding between glucose oxidase and composite was confirmed by ATR technique. The electrochemical experiments were done in 100 mM phosphate buffer at pH 7 and temperature of 25 °C with three electrodes including Ag/AgCl, platinum wire and the modified GCE as the reference electrode, the auxiliary electrode and working electrode respectively. The electrochemical results confirmed the activity and direct electron transfer of immobilized enzyme. The immobilized electroactive GOx concentration was estimated 3.06 × 10⁻¹¹ mol cm⁻². The results showed the immobilized enzyme had a good stability and maintained 90% of its performance after two weeks. The nanocomposite bioanode in an air-birthing biofuel cell and 100 mM glucose concentration showed 176 μWcm⁻² Power density. This strategy could be used for GOx-based biofuel cells.     

Electricity from landfill leachate using microbial fuel cells: Comparison with a biological aerated filter

Four experimental columns were employed in this study to investigate their performance under wastewater treatment conditions. One column was set-up as a biological aerated filter and the remaining three were set-up as microbial fuel cells (MFCs), two of which were connected to an external load whereas the third was left open circuit. The performance of the columns under several flow rates and leachate strengths was studied in terms of BOD₅ removal efficiencies and electricity generation, when a fixed resistive load was connected. Results obtained demonstrated that it is possible to generate electricity and simultaneously treat landfill leachate in MFC columns. Energy generation in MFC columns improved with increasing flow rates from 24 to 192 mL/h, while BOD₅ removal efficiency levels reached a maximum at 48 mL/h and dropped to relatively low values at higher flow rates. The maximum removal efficiencies were obtained at a loading rate of 0.81 kg BOD₅/m³ d for columns C1, C2 and C4 and 1.81 kg BOD₅/m³ d for column C3. Electrical output levels and BOD₅ concentrations at the MFC columns showed a linear relationship, which allows the system to be used as a BOD₅ sensor. Part of the BOD removal was not associated with power generation and was attributed to the presence of alternative end terminal electron acceptors and volatilisation. The MFC columns could reach the same or even higher removal efficiencies than those from the biological aerated filter with the advantage of producing energy and saving cost of aeration. To the best of the authors’ knowledge, this is the first study that compares the MFC technology with other conventional treatment systems for removing pollutants from wastewater.     

Effect of process change from perfusion to fed-batch on product comparability for biosimilar monoclonal antibody

A fed-batch process for the production of biosimilar monoclonal antibody was developed. Since the brand product is produced by perfusion process, the impact of process change from perfusion to fed-batch on product quality and cell performance was evaluated. Perfusion culture was performed at 0.47–1.00 (v/v/d) perfusion rate by spin-filter method with 15–17 μm mesh. Culture parameters such as pH (6.8–7.2), dissolved oxygen (40–70% air saturation), temperature (37 °C) and agitation speed (250 rpm) were applied in both culture modes. In terms of cell performance, volumetric productivity increased 3.7 times while process performance increased 7.5 times in fed-batch culture due to 10 times higher scalability. Considering the glycosylation pattern and charge variants, no significant changes in product quality were observed upon process change, although intact IgG level slightly decreased in fed-batch mode. The change of production media showed more effect on glycosylation patterns than the operation in different culture modes. Furthermore, there were no differences in biological activity, including TNFα, FcγRIIIa, and C1q-binding affinity. Through a scale-up study from 3 L to 12,500 L, it was confirmed that cell performance and product quality could be maintained. In conclusion, product quality of the fed-batch process was comparable to that of the reference product.     

Histochemical localization and characterization of chalcones on the foliar surface of Zuccagnia punctata Cav. Insights into their physiological role

Dihydroxychalcones as well as other metabolites synthesized via the phenylpropanoid pathway have a wide range of biological activities. Although this class of phenolic compounds is found in very large amounts in some tissues, their physiological significance remains unclear.This approach focused on the chemical analysis of Zuccagnia punctata leaf rinse extract in which dihydroxychalcones (99.25 μg 2′,4′-dihydroxychalcone/cm² and 73.38 μg 2′,4′-dihydroxy-3′-methoxychalcone/cm²) are the main constituents. Histochemical analysis (fluorescence microscope and emission scanning electron microscope coupled with an energy dispersive X-ray spectrometer) revealed a high flavonoid concentration on the foliar surface. The high accumulation of phenolic compounds, flavonoids or chalcones in the cuticle of Z. punctata leaves would act as defense mechanisms against UV radiation for the protection of photosynthetic tissues against oxidative stress.     

Genetic parameters for body weight,longissimus muscle depth and fat depth for Suffolk sheep in the Czech Republic

In this study, heritabilities and (co)variance components for body weight at 100 days (BW), muscle depth (MD) and fat depth (FD) were estimated for Suffolk, the most common sheep breed in the Czech Republic. Data from 1996 to 2004 were extracted from the sheep recording database of the Czech Sheep and Goat Breeding Association. Genetic parameters were estimated using multivariate animal models, including both direct and maternal genetic effects and permanent environmental effects. Average values for BW, MD and FD were 27.91 kg, 25.5 mm and 3.3 mm, respectively. Direct and maternal heritability for BW were 0.17 and 0.08, respectively, and direct heritabilities were 0.16 for MD and 0.08 for FD. Maternal heritability estimates for ultrasonic measurements were generally low. Direct genetic correlations between BW and MD and maternal genetic correlations between BW and MD were positive and favourable. Both direct genetic correlations between BW and FD and maternal genetic correlations between BW and FD were negative, but not significantly different from zero. The favourable genetic correlations between BW and MD make ultrasound measurements a valuable tool in breeding programs focusing on growth and carcass characteristics.     

Chromate reduction by resting cells of Achromobacter sp. Ch-1 under aerobic conditions

Chromate reduction was carried out by resting cells of Achromobacter sp. Ch-1 with lactate as electron donor under aerobic conditions. The reduction activity of the samples supplemented with lactate was two times as those without lactate. The reduction rate was influenced by initial pH and lactate concentration. Under the optimal conditions, pH 9.0 and 4000 mg l⁻¹ lactate supplement, reduction rate was 5.45 mg l⁻¹ min⁻¹. The reduction rate decreased with increasing of Cr(VI) concentrations and increased with cell densities proportionally. The maximum reduction limit of Ch-1 cells was obtained at 2107 mg l⁻¹ of Cr(VI).     

Measurement of plasma hydrogen sulfide in vivo and in vitro

The gasotransmitter hydrogen sulfide is known to regulate multiple cellular functions during normal and pathophysiological states. However, a paucity of concise information exists regarding quantitative amounts of hydrogen sulfide involved in physiological and pathological responses. This is primarily due to disagreement among various methods employed to measure free hydrogen sulfide. In this article, we describe a very sensitive method of measuring the presence of H₂S in plasma down to nanomolar levels, using monobromobimane (MBB). The current standard assay using methylene blue provides erroneous results that do not actually measure H₂S. The method presented herein involves derivatization of sulfide with excess MBB in 100 mM Tris–HCl buffer (pH 9.5, 0.1 mM DTPA) for 30 min in 1% oxygen at room temperature. The fluorescent product sulfide-dibimane (SDB) is analyzed by RP-HPLC using an eclipse XDB-C18 (4.6 × 250 mm) column with gradient elution by 0.1% (v/v) trifluoroacetic acid in acetonitrile. The limit of detection for sulfide-dibimane is 2 nM and the SDB product is very stable over time, allowing batch storage and analysis. In summary, our MBB method is suitable for sensitive quantitative measurement of free hydrogen sulfide in multiple biological samples such as plasma, tissue and cell culture lysates, or media.