Separation Buffer Research Articles

An Approach to Study Shape-Dependent Transcriptomics at a Single Cell Level.

Different types of cardiac hypertrophy have been associated with an increased volume of cardiac myocytes (CMs), along with changes in CM morphology. While the effects of cell volume on gene expression are well known, the effects of cell shape are not well understood. This paper describes a method that has been designed to systematically analyze the effects of CM morphology on gene expression. It details the development of a novel single-cell trapping strategy that is then followed by single-cell mRNA sequencing. A micropatterned chip has also been designed, which contains 3000 rectangular-shaped fibronectin micropatterns. This makes it possible to grow CMs in distinct length:width aspect ratios (AR), corresponding to different types of heart failure (HF). The paper also describes a protocol that has been designed to pick up single cells from their pattern, using a semi-automated micro-pipetting cell picker, and individually inject them into a separate lysis buffer. This has made it possible to profile the transcriptomes of single CMs with defined geometrical morphotypes and characterize them according to a range of normal or pathological conditions: hypertrophic cardiomyopathy (HCM) or afterload/concentric versus dilated cardiomyopathy (DCM) or preload/eccentric. In summary, this paper presents methods for growing CMs with different shapes, which represent different pathologies, and sorting these adherent CMs based on their morphology at a single-cell level. The proposed platform provides a novel approach to high throughput and drug screening for different types of HF.

Sensitive analysis of doxorubicin and curcumin by micellar electromagnetic chromatography with a double wavelength excitation source.

Doxorubicin has been extensively used to treat cancers, and there are recent findings that the anticancer activities can be enhanced by curcumin. Although the two compounds have native fluorescence, they can hardly be quantified directly simultaneously using the laser-induced fluorescence (LIF) detection method. To avoid complex fluorescence derivatization and introduction of interfering components, a highly sensitive double wavelength excitation source LIF (D-W-Ex-LIF) detector composed of a 445-nm and 488-nm commercial laser diode was constructed to detect them simultaneously. Rhodamine 6G was selected as an internal standard, because its fluorescence can be excited at 445nm and 488nm. The native fluorescence of doxorubicin and curcumin and their resolution were enhanced by introducing mixed micelles. The optimal electrophoretic separation buffer was 10mM borate buffer containing 20mM Triton X-100, 5mM sodium dodecyl sulfate, and 30% (v/v) methanol at pH9.00. Therefore, the developed method was specific, accurate, and easily operable. Its limits of detection for doxorubicin and curcumin in human urine samples were 4.00 × 10-3 and 1.00 × 10-2μg/mL, respectively, and the limits of quantification were 1.00 × 10-2 and 3.00 × 10-2μg/mL, respectively. The recoveries were 94.9-109.1%. Graphical abstract.

Energy-Efficient Dual-Hop Internet of Things Communications Network With Delay-Outage Constraints

This article considers a dual-hop Internet of Things communications network where sensor nodes transmit data to a gateway either directly or via other nodes using dual-hop communications. Each node employs separate transmission buffers to store its own sensing data and data received from other nodes. End-to-end delay quality-of-service constraints in terms of the maximum acceptable delay-outage probabilities are imposed. We investigate energy-efficient adaptive resource allocation problems (i.e., joint link scheduling, rate, and power allocation) to support minimum data rates of the nodes. A novel approach is proposed exploiting asymptotic delay analysis to first determine the achieved delay exponents of the queue length tail distributions to satisfy the delay-outage constraints. Next, the relation between the delay exponents and resource allocation variables are derived. Last, the solutions to the resulting constrained optimization problems are obtained using the Lagrangian approach and convex optimization. Illustrative examples demonstrate the effects of the rate requirements and delay constraint stringency on the power consumption and routing configuration.

Defining separation zones for coastal birds at a wetland of global importance

Abstract ContextThe disruption of normal activities by humans (i.e. ‘anthropogenic disturbance’) can have important behavioural, physiological and population effects on coastal birds. These negative effects include increased vigilance, increased energy expenditure and reduced nesting success. To overcome this, separation distances (e.g. buffers and setbacks) are often used to separate threatening stimuli, such as humans, from wildlife. However, in most instances the determination of separation distances are based on little empirical information. This is particularly true for Africa, which supports huge populations of Palearctic migrant shorebirds. AimTo determine suitable separation distances that will reduce anthropogenic disturbance to the coastal bird community at West Coast National Park, South Africa. MethodsThe distance at which a behavioural response (i.e. flight initiation distance, FID) occurred among 15 common coastal bird species when presented with an approaching human was measured. Linear mixed-effects models were used to determine the relationship between FID and body size, migratory status, intraspecific flock size, flock species richness, foraging behaviour and several environmental covariates. Key resultsBody mass was significantly and positively correlated with FID, indicating that larger birds are more sensitive to the anthropogenic stimulus. Furthermore, it was shown that migratory birds had longer FIDs, suggesting that they are less risk tolerant compared with resident birds. The distance at which the approach was initiated (i.e. the start distance, SD) was also significantly and positively correlated with FID, supporting the hypothesis that birds will flush early to avoid the potential negative consequences of fleeing too late. Finally, there was a novel significant effect of increasing wind speed on increasing FID, suggesting a thermoregulatory trade-off. There was no effect of foraging behaviour, flock size or flock composition on FID. ConclusionBased on these data, buffer distances for tidal habitats at West Coast National Park should be set at ~170 m. ImplicationsImproved understanding of FID, and the morphological and behavioural predictors thereof, will enable West Coast National Park and other protected areas to designate separation distances or buffer zones that reduce the negative effects of anthropogenic disturbance on shorebird communities.

Assessment and Comparison of the Overall Analytical Potential of Capillary Electrophoresis and High-Performance Liquid Chromatography Using the RGB Model: How Much Can We Find Out?

The choice between capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) is not easy and depends on many factors. An attempt to facilitate this choice is this work, in which both techniques have been confronted on the basis of the RGB model, offering a transparent and pictorial way to compare individual parameters as well as the overall analytical potential of each tool.To ensure the universal nature of the comparison, a simple in composition and chemically diverse model sample was used, accompanied by the data processing method reducing the potential impact of analyte selection. Moreover, permanent coating of the inner surface of the capillary and addition of a surfactant to the separation buffer were considered as the additional factors that may affect the assessment of the CE technique. The presented analysis can be valuable in any discussions about the intrinsic advantages and disadvantages of CE and HPLC, and divagations on how they affect the overall potential and usability of each. We also provide access to the Excel worksheets used for the assessment, which can be easily modified to reevaluate the methods with a different selection of variables, and analyze other possible scenarios.

Bridging the gap between visual temporary memory and working memory: The role of stimuli distinctiveness.

Visual working memory (VWM) is traditionally assumed to be immune to proactive interference (PI). However, in a recent study (Endress & Potter, 2014), performance in a visual memory task was superior when all items were unique and hence interference from previous trials was impossible, compared to a standard condition in which a limited set of repeating items was used and stimuli from previous trials could interfere with the current trial. Furthermore, when all the items were unique, the estimated memory capacity far exceeded typical capacity estimates. Consequently, the researchers suggested the existence of a separate memory buffer, the "temporary memory," which has an unbounded capacity for meaningful items. However, before accepting this conclusion, methodological differences between the repeated-unique procedure and typical estimates of VWM should be considered. Here, we tested the extent to which the exceptional set of heterogeneous, complex, meaningful real-world objects contributed to the large PI in the repeated-unique procedure. Thus, the same paradigm was employed with a set of real-world objects and with homogenous sets (e.g., houses, faces) in which the items were meaningful, yet less visually distinct, and participants had to rely on subtle visual details to perform the task. The results revealed a large PI effect for real-world heterogeneous objects, but substantially smaller effects for the homogenous sets. These findings suggest that there is no need to postulate a new memory buffer. Instead, we suggest that VWM capacity and vulnerability to PI are highly influenced by task characteristics, and specifically, by the stimuli distinctiveness. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Professional or Amateur? The Phonological Output Buffer as a Working Memory Operator.

The Phonological Output Buffer (POB) is thought to be the stage in language production where phonemes are held in working memory and assembled into words. The neural implementation of the POB remains unclear despite a wealth of phenomenological data. Individuals with POB impairment make phonological errors when they produce words and non-words, including phoneme omissions, insertions, transpositions, substitutions and perseverations. Errors can apply to different kinds and sizes of units, such as phonemes, number words, morphological affixes, and function words, and evidence from POB impairments suggests that units tend to substituted with units of the same kind—e.g., numbers with numbers and whole morphological affixes with other affixes. This suggests that different units are processed and stored in the POB in the same stage, but perhaps separately in different mini-stores. Further, similar impairments can affect the buffer used to produce Sign Language, which raises the question of whether it is instantiated in a distinct device with the same design. However, what appear as separate buffers may be distinct regions in the activity space of a single extended POB network, connected with a lexicon network. The self-consistency of this idea can be assessed by studying an autoassociative Potts network, as a model of memory storage distributed over several cortical areas, and testing whether the network can represent both units of word and signs, reflecting the types and patterns of errors made by individuals with POB impairment.

Cyclodextrin-micellar electrokinetic chromatography of apolipoproteins on human very low-density lipoprotein.

The apolipoproteins (APOs) of human very low-density lipoprotein (VLDL) were investigated by an optimized cyclodextrin-micellar electrokinetic chromatography (CD-MEKC) method. The separation buffer consisted of 20mM sodium phosphate, 40mM bile salts (50% sodium cholate and 50% sodium deoxycholate), 25mM carboxymethyl-β-cyclodextrin (CM-β-CD) (pH 7.0). For CD-MEKC separation, a sample injection time of 12 s, a separation voltage of 15KV, and a capillary temperature of 15°C were chosen. The optimal CD-MEKC method showed good resolution and repeatability for VLDL APOs. Identification and quantitation of VLDL APOs CI, CIII, and E were based on comparison with human APO standards. Good linear relationships with correlation coefficient (R2 ) 0.99 were obtained for APOs CI, CIII, and E standards. For these three APOs, the linear ranges were within 0.01-0.54mg/mL, and the concentration limits of detection (LODs) were lower than 0.02mg/mL. Moreover, VLDL APOs from four uremic patients and four healthy subjects were compared. The uremic and healthy CD-MEKC profiles showed dramatic difference. The levels of APO CIII were significantly higher for two patients, and the level of APO E was significantly higher for one patient. This study might be helpful for following the disease development of uremia and cardiovascular disease (CVD) in the future.

Enantiomeric separation of homocysteine and cysteine by electrokinetic chromatography using mixtures of γ-cyclodextrin and carnitine-based ionic liquids

Two new chiral ionic liquids based on the ester of the non-protein amino acid carnitine as cationic counterpart were synthesized, characterized and evaluated as chiral selectors for the enantiomeric separation of homocysteine and cysteine derivatized with 9-fluorenylmethoxycarbonyl chloride by electrokinetic chromatography. The use of both ionic liquids, l-carnitine methyl ester bis(trifluoromethane)sulfonimide and l-carnitine methyl ester l-(+)-lactate, as sole chiral selectors in the separation buffer, did not allow the enantiomeric separation of the studied amino acids. However, the combined use of l-carnitine methyl ester bis(trifluoromethane)sulfonimide ionic liquid with γ-CD forming a dual system led to the enantiomeric separation of both analytes and showed the existence of a strong synergistic effect. On the contrary, the dual system γ-CD plus l-carnitine methyl ester l-(+)-lactate did not improve the enantiomeric separations with respect to those obtained using the CD alone. The influence of different experimental variables such as buffer composition and pH, and ionic liquid concentration was investigated. Also, the nature of the anionic moiety was evaluated by comparing the results obtained with both ionic liquids when combined with γ-CD. The use of 2 mM γ-CD combined with 5 mM l-carnitine methyl ester bis(trifluoromethane)sulfonimide in a 50 mM phosphate buffer at pH 7.0 enabled to achieve the simultaneous enantiomeric separation of homocysteine and cysteine with high resolution values (>6.0) in analysis times close to 12 min.

Kinetic analysis of substrate competition in enzymatic reactions with β-D-galactosidase by capillary electrophoresis / dynamic frontal analysis

Competitive inhibition between two substrates with an enzyme is investigated by capillary electrophoresis/dynamic frontal analysis (CE/DFA). Enzymatic hydrolyses of o-nitrophenyl β-D-galactopyranoside and p-nitrophenyl β-D-galactopyranoside with β-D-galactosidase were examined as a model competitive reaction. A sample solution containing the two substrates was injected into a capillary filled with a separation buffer containing an enzyme. Enzymatic hydrolysis occurred during the electrophoresis, and the products of o-nitrophenol and p-nitrophenol were continuously formed and resolved from the sample zone. Two-steps plateau signal was detected with the two-substrate solutions based on the difference in the effective electrophoretic mobility of o-nitrophenol and p-nitrophenol. Michaelis-Menten constants and inhibition constants were determined with the plateau heights. Usefulness of CE/DFA on competitive inhibition analysis is demonstrated in this study.

Capillary Electrophoretic Characterization of Carbon Nanodots Prepared from Glutamic Acid in an Electric Furnace

Carbon nanodots (CNDs) prepared from glutamic acid or glutathione in an electric furnace were characterized by capillary electrophoresis. Two major peaks were detected in the electropherograms by capillary zone electrophoresis, corresponding to anionic and less-charged CNDs. The effective electrophoretic mobility of the anionic CND formed from glutamic acid was almost identical over neutral to weakly alkaline pH range, and the CND would not contain significant amount of amino group. On the other hand, the effective electrophoretic mobility tended to decrease with decreasing pH at weakly acidic pH conditions, suggesting the functional groups of carboxylate moiety on the anionic CNDs. Dodecyl sulfate ion was added in the separation buffer to give anionic charge to the less-charged CND by adsorption. However, the anionic charge induced was little, and the dodecyl sulfate ion was not likely adsorbed on the less-charged CND and the CND would be hydrophilic.

Determination of acid dissociation constants of flavin analogues by capillary zone electrophoresis.

Acid dissociation constants (pKa ) of nine kinds of flavin analogues as molecular catalyst candidates were determined by CZE. Although some of the analogues are instable and degradable under the light exposure or in alkaline aqueous solutions, the effective electrophoretic mobility of the flavin analogue of interest has been measured with the residual substance. The pKa values of the flavin analogues were analyzed through the changes in the effective electrophoretic mobility with varying pH of the separation buffer. One or two steps pKa values were determined by the analysis. One of the degraded species from the flavin analogues, lumichrome, was also detected in the CZE analysis, and its pKa values were also determined. While coexisting impurities generated over the storage conditions were found in some analogues, the pKa values of the target analogues were successfully determined with the help of the CZE separations. A pressure-assisted CZE was utilized for the determination or the estimation of the pKa values of such analogues as possessing carboxylic acid moiety.

Long alkyl chain-grafted carbon nanotube-decorated binary-core phase-change microcapsules for heat energy storage: Synthesis and thermal properties

Microencapsulated phase-change materials (MPCMs) with carbon nanotube (CNT)-enhanced fillers and binary cores, which exhibit improved thermal conductivity and an adjustable working temperature range, are highly desirable for various heat energy storage applications. However, degraded properties are usually found for the CNT-enhanced MPCMs due to the poor compatibility of CNTs with PCMs, and the existing types of binary-core MPCMs (BCMPCMs) are quite limited. Herein, we prepared a novel type of BCMPCM with a n-octadecane/n-octacosane (C18/C28) mixture as the core, octadecyl isocyanate-grafted CNTs (OICNTs) as the enhancers and melamine-formaldehyde (MF) resin as the shell via in situ polymerization. Morphological and structural characterization of the OICNT-enhanced BCMPCMs demonstrated uniform distributions of OICNTs in both the core and shell. The determined phase-change temperatures were approximately 26 °C and 43 °C, with small degrees of supercooling. The phase-change enthalpy was as high as 187.9 J/g for a relatively large OICNT loading (3 wt%). Thermal conductivity measurements of the OICNT-enhanced BCMPCM/epoxy resin composites showed a 71.4% increase over that of the pure BCMPCM/epoxy resin composite. Moreover, the BCMPCMs experienced two separate temperature buffer stages resulting from the phase-change processes and exhibited a prominent thermal regulation capacity, which is of significance for their potential applications in the cooling of electronic devices, battery thermal management, utilization of waste heat, and air conditioning systems, etc.

Correlation minimizing replay memory in temporal-difference reinforcement learning

Online reinforcement learning agents are now able to process an increasing amount of data which makes their approximation and compression into value functions a more demanding task. To improve approximation, thus the learning process itself, it has been proposed to select randomly a mini-batch of the past experiences that are stored in the replay memory buffer to be replayed at each learning step. In this work, we present an algorithm that classifies and samples the experiences into separate contextual memory buffers using an unsupervised learning technique. This allows each new experience to be associated to a mini-batch of the past experiences that are not from the same contextual buffer as the current one, thus further reducing the correlation between experiences. Experimental results show that the correlation minimizing sampling improves over Q-learning algorithms with uniform sampling, and that a significant improvement can be observed when coupled with the sampling methods that prioritize on the experience temporal difference error.

Capillary Electrophoretic Characterization of Water-soluble Carbon Nanodots Formed from Glutamic Acid and Boric Acid under Microwave Irradiation.

Water-soluble carbon nanodots (CND) were synthesized under microwave irradiation from glutamic acid or glutamic acid-boric acid mixture. The CNDs were collected in an aqueous solution through size fractionation by centrifugal filtration. The CNDs thus prepared were subjected to characterization by capillary electrophoresis (CE). A peak signal of anionic substance was detected in the electropherogram, and it was found to be a major component of the CNDs. The effective electrophoretic mobility of the major component was almost identical over the pH range between 6.7 and 11.6, suggesting that the functional group of amine or boric acid moiety was not included in the CNDs. The effective electrophoretic mobility decreased at an acidic pH of less than 5, and it was suggested that carboxylate moiety was included in the CNDs. A signal of less-charged CNDs was also detected in the electropherogram, and the CNDs were characterized by a CE format of micellar electrokinetic chromatography. Two or four peaks were detected just after the electroosmotic flow; the less-charged CNDs were thus hydrophilic. The affinity interaction was also examined between the major anionic CNDs and a hydrophobic pairing cation. The peak signal of the major anionic CNDs broadened, and its theoretical number of plates decreased in the presence of tetrabutylammonium ion in the separation buffer. A small portion of the anionic CNDs were a little hydrophobic at different degrees, and their effective electrophoretic mobility decreased by the hydrophobic interaction, resulting in peak broadening of the anionic CNDs.

A platform for studying the transfer of Chlamydia pneumoniae infection between respiratory epithelium and phagocytes

The obligate intracellular bacterium, Chlamydia pneumoniae, has been identified as a risk factor for several chronic inflammatory diseases in addition to respiratory tract infections. The dissemination of C. pneumoniae from respiratory tract to secondary sites of infection occurs via infected monocyte / macrophage line cells, in which C. pneumoniae can persist as an antibiotic-refractory phenotype. To allow more detailed studies on the epithelium-monocyte/macrophage transition of the infection, new in vitro bioassays are needed. To this end, a coculture system with human continuous cell lines was established. Respiratory epithelial HL cells were infected with C. pneumoniae and THP-1 monocytes were added into the cultures at 67 h post infection. After a 5 h coculture, THP-1 cells were collected with a biotinylated HLA antibody and streptavidin-coated magnetic beads and C. pneumoniae genome copy numbers in THP-1 determined by quantitative PCR. The assay was optimized for cell densities, incubation time, THP-1 separation technique and buffer composition, and its robustness was demonstrated by a Z' value of 0.6. The mitogen-activated protein kinase (MAPK) inhibitors: SP600125 (JNK inhibitor), SB203580 (p38 inhibitor) and FR180204 (ERK inhibitor) suppressed the transfer of C. pneumoniae from HL to THP-1 cells, making them suitable positive controls for the assay. Based on analysis of separate steps of the process, the MAPK inhibitors suppress the bacterial entry to THP-1 cells. The transfer of C. pneumoniae from epithelium to phagocytes represents a crucial step in the establishment of persistent infections by this pathogen, and the presented methods enables future studies to block this process by therapeutic means.

Can phase separation buffer cellular noise?

By suppressing concentration fluctuations, condensation may stabilize cellular processes

Capillary Electrophoresis/Dynamic Frontal Analysis for the Enzyme Assay of 4-Nitrophenyl Phosphate with Alkaline Phosphatase

A substrate of 4-nitrophenyl phosphate was enzymatically hydrolyzed by alkaline phosphatase (ALP) in a capillary tube, while an injected zone of the substrate was electrophoretically migrating in the separation buffer containing the enzyme by capillary electrophoresis (CE). During CE migration of the substrate from the start time of the electrophoresis to the detection time of the substrate, the substrate was continuously hydrolyzed by ALP to form a product of 4-nitrophenolate, and a plateau signal of 4-nitrophenolate was detected as a result of the zero-order kinetic reaction. The height of the plateau signal was directly related to the reaction rate, and it was used for the determination of a Michaelis-Menten constant through Lineweaver-Burk plots. Since the plateau signal is attributed to the dynamic formation of the product by the enzymatic reaction in CE, this analysis method is named as capillary electrophoresis/dynamic frontal analysis (CE/DFA). In CE/DFA, the CE separation is included on detecting the plateau signal, and the hydrolysis product before the sample injection is resolved from the dynamically and continuously formed product. The inhibition of the enzyme with the product is also eliminated in CE/DFA by the CE separation.

Kinetic analysis of an enzymatic hydrolysis of p-nitrophenyl acetate with carboxylesterase by pressure-assisted capillary electrophoresis/dynamic frontal analysis.

An enzymatic hydrolysis of p-nitrophenyl acetate with carboxylesterase was analyzed by capillary electrophoresis/dynamic frontal analysis (CE/DFA). A plateau signal was expected with the anionic product of p-nitrophenol by the CE/DFA applying in-capillary reaction and the continuous CE resolution of the product from the substrate zone. However, the plateau height was not sufficient, and/or the plateau signal fluctuated and drifted. Therefore, a pressure assist was utilized in the CE/DFA to detect the product zone fast and to average the fluctuated plateau signal by mixing in a laminar flow. The plateau signal became relatively flat and its height was developed by the pressure-assisted capillary electrophoresis/dynamic frontal analysis (pCE/DFA). The plateau height was used for the Michaelis-Menten analysis, and a Michaelis-Menten constant was determined as KM = 0.83 mmol L-1. An enzyme inhibition was also examined with bis(p-nitrophenyl) phosphate by adding it in the separation buffer. The height of the plateau signal decreased by the inhibition, and a 50% inhibitory concentration was determined as IC50 = 0.79 μmol L-1. The values of KM and IC50 obtained in this study agreed well with the reported values. Since the proposed pCE/DFA includes electrophoretic migration of the substrate zone in a capillary, it is also noticed that the deactivation of the enzyme by ethanol on the preparation of the substrate solution can be avoided, as well as the exclusion of the inhibition by the product.

Differentiated impacts of environmental policies on the Colombian Frontier: coercive conservation and containment of illicit activities in the Pacific and the Ariari region

ABSTRACT This article, through the use of political ecology perspectives on coercive conservation, aims to explain how in two separate Colombian Natural Parks and buffer zones, environmental policies designed to (re)take control of the frontier, have produced a similar territorial differentiation in the contention of illicit activities. Los Farallones in the Colombian Pacific and La Macarena/Puerto Rico in the Ariari region have experienced different stages of the armed conflict and are at the center of this analysis. I argue that in the contexts of both conflict escalation (1998–2007) and conflict de-escalation (2008–2016), the State in its attempt to control the frontier has not only had military intervention in areas of conservation but has also reinforced environmental programs that attack illegal mining and coca, producing both a territorially differentiated containment of illicit activities and an uneven progression of the illicit frontier.