Reconstitution Technique Research Articles

Structure-based validation of recombinant light-harvesting complex II.

Light-harvesting complex II (LHCII) captures sunlight and dissipates excess energy to drive photosynthesis. To elucidate this mechanism, the individual optical properties of pigments in the LHCII protein must be identified. In vitro reconstitution with apoproteins synthesized by Escherichia coli and pigment-lipid mixtures from natural sources is an effective approach; however, the local environment surrounding each pigment within reconstituted LHCII (rLHCII) has only been indirectly estimated using spectroscopic and biochemical methods. Here, we used cryo-electron microscopy to determine the 3D structure of the rLHCII trimer and found that rLHCII exhibited a structure that was virtually identical to that of native LHCII, with a few exceptions: some C-terminal amino acids were not visible, likely due to aggregation of the His-tags; a carotenoid at the V1 site was not visible; and at site 614 showed mixed occupancy by both chlorophyll a and b molecules. Our observations confirmed the applicability of the in vitro reconstitution technique.

Analysis of reconstitution process, stability, stability of intravenous antibiotics in hospitalised patients in a private hospital

Introduction: reconstitution of sterile preparations is a series of changes in the drug from its original condition to a new product by dissolving or adding other ingredients carried out aseptically by the pharmacy. Aim: determine the suitability of the reconstitution technique, the suitability of stability (solvent type and solvent volume), and the suitability of the sterility of the reconstitution product. Methods: data collection obtained by observation is carried out with the help of a checklist that is adapted to hospital standard operating procedures (SOPs), Inject-able Drug Guide, guidelines for mixing inject-able drugs, and preparations. 48 processes of reconstitution of intravenous antibiotics were carried out in hospitals that could be observed as samples. Results: conformity to the preparation stage were 99%, the mixing stage was 73%, the labelling was 100%, and the labelling was 90%. In evaluating the stability of the solvent type category, the suitability of 100%. In the volume category of the solvent used, 90% were suitable, and 10% were unsuitable for the antibiotic’s cefotaxime 1 gram, vancomycin 1 gram, and sulbactam + ampicillin Na 1 gram. From the sterility test results, the reconstitution of ceftriaxone intravenous antibiotic preparations showed that the bacterial preparations were free of microorganisms (sterile). Conclusion: the personnel performing the reconstitution are nurses trained in intravenous antibiotic recovery. One of the factors that caused the reconstitution process not to be under the hospital's KSB was the lack of training and supervision from the hospital management. SPO recovery is not installed in an intravenous drug mixing room.

Split Luciferase-Fragment Reconstitution for Unveiling RNA Localization and Dynamics in Live Cells.

The intracellular distribution and dynamics of RNAs play pivotal roles in various physiological phenomena. The ability to monitor the amount and localization of endogenous RNAs in living cells allows for elucidating the mechanisms of various intracellular events. Protein-based fluorescent RNA probes are now widely used to visualize and analyze RNAs in living cells. However, continuously monitoring the temporal changes in RNA localization and dynamics in living cells is challenging. In this study, we developed a bioluminescent probe for spatiotemporal monitoring of RNAs in living cells by using a split-luciferase reconstitution technique. The probe consists of split fragments of a bioluminescent protein, NanoLuc, connected with RNA-binding protein domains generated from a custom-made mutation of a PUM-HD. The probe showed rapid luminescence intensity changes in response to an increase or decrease in the amount of a target RNA in vitro. In live-cell imaging, temporal alteration of the intracellular distribution of endogenous β-actin mRNA was visualized in response to extracellular stimulation. Furthermore, the application of the probe to the visualization of the specific localization of β-actin mRNA in primary hippocampal neurons was conducted. These results demonstrate the capability of the bioluminescent RNA probe to monitor the changes in localization, dynamics, and the amount of target RNA in living cells.

Regulatory mechanisms of mitochondrial calcium uptake by the calcium uniporter complex.

Mitochondria play an important role in energy conversion as well as in intracellular calcium (Ca2+) storage. Ca2+ uptake from the cytosol to the mitochondria is mediated by the calcium uniporter, which functions as a Ca2+ ion channel. However, the molecular composition of this uniporter has remained unclear until recently. The Ca2+ ion channel consists of seven subunits. The yeast reconstitution technique revealed that the mitochondrial calcium uniporter (MCU) and essential MCU regulatory element (EMRE) are the core subunits of the complex. Furthermore, detailed structure-function analyses of the core subunits (MCU and EMRE) were performed. In this review, the regulatory mechanism of mitochondrial Ca2+ uptake is discussed.

Visualizing Actin and Microtubule Coupling Dynamics <em>In Vitro</em> by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy.

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Study on Data Multiplication of Impact Toughness by Specimen Reconstitution Technique and Master Curve

Study on Data Multiplication of Impact Toughness by Specimen Reconstitution Technique and Master Curve

Microbial Diversity of Reconstituted, Degraded, and Agricultural Soils Assessed by 16S rDNA Multi-Amplicon Sequencing

The microbial diversity is, among soil key factors, responsible for soil fertility and nutrient biogeochemical cycles, and can be modified upon changes in main soil physicochemical properties and soil pollution. Over the years, many restoration techniques have been applied to restore degraded soils. However, the effect of these approaches on soil microbial diversity is less understood and thus requires more investigation. In this study, we analyzed the impact, on soil microbial diversity of a patented novel technology, used to restore degraded soils. Soil samples were collected from three nearby sites located in Borgotrebbia, Piacenza, Italy, and categorized as reconstituted, degraded, and agricultural soils. After total soil DNA extraction, 16S rDNA multi-amplicon sequencing was carried out using an Ion GeneStudio S5 System to compare soils’ bacterial community profiles. Sequenced reads were processed to assign taxonomy and then key microbial community differences were identified across the sampling sites. Species diversity featured significant abatement at all rank levels in the degraded soil when compared to the agricultural control. The 5 year restoration technique showed full recovery of this index at the genus level but not at the phylum level, displaying a rank-dependent gradient of restored richness. In parallel, the abundance of genes involved in the nitrogen (N) biogeochemical cycle was assessed using quantitative Real-Time PCR (qPCR). Total DNA content was significantly higher (p < 0.05) in degraded (μ = 12.69 ± 2.58 μg g−1) and reconstituted (μ = 11.73 ± 1.65 μg g−1) soil samples when compared to the agricultural soil samples (μ = 2.39 ± 0.50 μg g−1). The taxonomic diversity of each soil site was significantly different, with some instances unique of the agricultural soil even at the phylum level. The analysis of N functional genes showed that the relative abundance of bacterial amoA (p < 0.05) and nosZ (p < 0.01) genes were significantly lower in the agricultural than in the reconstituted and degraded soils. We concluded that the application of the soil reconstitution technique appears to enhance the active microbial community, with distinct diversity and functionality towards genes involved in N biogeochemical cycle, as compared to both the degraded and the agricultural soil.

Impact of Type of Nebulizers on Liposomes Prepared by Bed Side Reconstitution Technique

Impact of Type of Nebulizers on Liposomes Prepared by Bed Side Reconstitution Technique

Elucidating the Mechanism Behind Sodium-Coupled Neurotransmitter Transporters by Reconstitution.

Sodium-coupled neurotransmitter transporters play a fundamental role in the termination of synaptic neurotransmission, which makes them a major drug target. The reconstitution of these secondary active transporters into liposomes has shed light on their molecular transport mechanisms. From the earliest days of the reconstitution technique up to today's single-molecule studies, insights from live functioning transporters have been indispensable for our understanding of their physiological impact. The two classes of sodium-coupled neurotransmitter transporters, the neurotransmitter: sodium symporters and the excitatory amino acid transporters, have vastly different molecular structures, but complementary proteoliposome studies have sought to unravel their ion-dependence and transport kinetics. Furthermore, reconstitution experiments have been used on both protein classes to investigate the role of e.g. the lipid environment, of posttranslational modifications, and of specific amino acid residues in transport. Techniques that allow the detection of transport at a single-vesicle resolution have been developed, and single-molecule studies have started to reveal single transporter kinetics, which will expand our understanding of how transport across the membrane is facilitated at protein level. Here, we review a selection of the results and applications where the reconstitution of the two classes of neurotransmitter transporters has been instrumental.

Extended Trochanteric Osteotomy Closure Performed Before or After Canal Preparation and Stem Impaction Does Not Affect Axial Stability

BackgroundAn extended trochanteric osteotomy (ETO) safely addresses femoral component removal during challenging revision total hip arthroplasty. However, no prior study has evaluated whether a difference in axial stability exists between ETO closure performed before (reconstitution) or after (scaffolding) canal preparation and stem impaction. We hypothesized that given the absence of clinical reports of outcome differences despite the wide use of both practices, no significant difference in the initial axial stability would exist between the 2 fixation techniques. MethodsETOs were performed and repaired using the reconstitution technique for the 6 right-sided femora and the scaffolding technique for the six left-sided femora. The 195-mm long, 3.5°-tapered splined titanium monobloc stems were impacted into 6 matched pairs of human fresh cadaveric femora. Three beaded cables were placed in a standardized fashion on each specimen, 1 for prophylaxis against osteotomy propagation during reaming/impaction and 2 to close the ETO. Stepwise axial loading was performed to 2600 N or until failure, which was defined as subsidence >5 mm or femoral/cable fracture. ResultsAll specimens successfully resisted axial testing, with no stem in either ETO repair group subsiding >2 mm. The mean subsidence for the reconstitution group was 0.9 ± 0.4 mm, compared to 1.2 ± 0.5 mm for the scaffolding group (P = .2). ConclusionIn this cadaveric model with satisfactory proximal bone stock, no difference existed between the reconstitution and scaffolding ETO repair techniques, and both provide sufficient immediate axial stability in a simulated revision total hip arthroplasty setting under physiologic loads.

Reissner’s Mixed Variational Theorem and triangular finite element discretizations: an energetic interpretation

Reissner’s Mixed Variational Theorem (RMVT) presents both displacements and transverse stresses as primary variables. This property allows the a-priori fulfillment of both interlaminar compatibility and equilibrium with potentially excellent numerical performance. However, the triangular finite element based on RMVT has never been assessed from an energetic perspective. This aspect is investigated in the present contribution for the first time: the functional reconstitution technique is extended to RMVT-based triangular elements (retaining transverse normal stress) and it is demonstrated that the elements exhibit significantly lower errors than the corresponding displacement-based formulation. Moreover, the percentage errors on the approximation of the semi-complementary energy is shown to be invariant with the plate thickness-to-width ratio.

The Molecular Mechanisms of Mitochondrial Calcium Uptake by Calcium Uniporter

Mitochondria play a role as intracellular calcium stores as well as energy conversion functions. Excessive calcium accumulation in mitochondria induces cell death and induces diseases such as ischemia-reperfusion injury. Mitochondrial calcium uptake is considered to be mediated by calcium uniporters, which have attracted much attention as potential drug targets. Although calcium uniporter was shown to function as an ion channel, the molecular mechanisms have long been unclear. In this decade, the molecular composition of the calcium uniporter complex was discovered; the calcium uniporter consists of the 7 subunits. Each subunit has no structural similarity to other Ca ion channels; thus, the novel molecular mechanism of the Ca2+ uptake by calcium uniporter is of interest. Although calcium uniporter is conserved in human to warm, yeast lack mitochondrial calcium uptake activity. In the previous study, various subunits of mammalian calcium uniporter were expressed in the yeast mitochondria. As a result, although the expression of each subunit alone did not affect on the mitochondrial calcium uptake activity, the co-expression of mitochondrial calcium uniporter (MCU) and essential MCU regulator (EMRE) enabled to reconstitute calcium uptake activity in yeast mitochondria. This indicated that MCU and EMRE are key factors of the calcium uptake activity in mitochondria. This yeast reconstitution technique has also enabled us to perform detailed structure-function analysis of the MCU and EMRE. In this paper, we will discuss the molecular mechanism of Ca2+ uptake and the prospects for drug discovery.

The Molecular Mechanisms of Mitochondrial Calcium Ion Channel, Calcium Uniporter

Mitochondria play a role as intracellular calcium stores as well as energy conversion functions. Mitochondrial calcium uptake is mediated by mitochondrial calcium ion channel, “calcium uniporter”. In this decade, the molecular composition of the calcium uniporter complex was discovered; the calcium uniporter consists of the 7 subunits. Using the yeast reconstitution technique, we showed that MCU and EMRE are core subunits of the complex. The yeast reconstitution technique has also enabled us to perform detailed structure-function analysis of the MCU and EMRE. In this review, we will discuss the molecular mechanism of mitochondrial Ca2+ uptake.

Functional Reconstitution of Reissner’s Mixed Variational Theorem for Finite Element Applications

The functional reconstitution technique is used for the first time to theoretically and a priori assess the numerical performance of Reissner’s Mixed Variational Theorem in the case of linear quadrilateral finite element, Generalized Unified Formulation, and composite materials. From the finite element discretization, the semi-complementary energy is reconstructed in integral form. Analytical expressions for the exact and spurious terms are obtained. Using the elasticity solution for a simply supported plate, it is numerically shown that these spurious contributions do not significantly alter the quality of the approximation of the total level of energy, with overall excellent computational performance. On the other hand, when the starting functional is the strain energy and the principle of virtual displacement is adopted, the finite-element-introduced additional terms and in particular the contributions associated to the transverse shear stresses are demonstrated to add a very large error, which is a priori proven to be eliminated with selective integration. The presented technique could be extended to analyze the performance of other finite elements (e.g., higher-order triangular ones) within Reissner’s Mixed Variational Theorem and Generalized Unified Formulation.

Quality assessment of a new in-mould slurry deposition method for triaxial specimen reconstitution of clean and silty sands

An innovative specimen reconstitution technique for sandy and silty soils that simulates underwater deposition is presented and evaluated. The technique is an upgraded version for triaxial testing of the well-established slurry deposition method. This novel setup integrates the reconstitution mould and the mixing tube into a single unit to avoid transferring the sample from the mixing tube to the mould. This subtle, but critical, modification enables reconstitution of very loose specimens as sample transfer disturbance, which can be significant, is eliminated. The quality of specimens prepared by the new reconstitution method was assessed by experiments on a clean sand from the UK (Ham River sand) and a silty sand from Norway (Øysand). The method, as any slurry-based procedure, is capable of producing homogeneous specimens with high initial degree of saturation, even in the absence of back pressure. The procedure is shown to be suitable for sands with or without fines. Moreover, the new method is able to achieve a wide range of initial void ratios, from very loose to very dense, without imposing any particle crushing in the latter case.

Getting GPCRs into liposomes

G protein-coupled receptors (GPCRs) are key drug targets and important signaling proteins embedded in cell membranes. To study the proteins’ specific signaling interactions more easily by eliminating the complexities of the cellular environment, researchers often use detergents to extract them from cell membranes and then reconstitute the receptors in synthetic membranes. The detergents can modify ligand binding interactions and other native properties of GPCRs, so researchers must use dialysis or adsorbents to remove detergents before studying the reconstituted proteins. A team led by Neal Devaraj and Roger Sunahara of the University of California, San Diego, developed a GPCR reconstitution technique that doesn’t require detergent cleanup (J. Am. Chem. Soc. 2017, DOI: 10.1021/jacs.6b12830). In the technique, called in situ lipid synthesis for protein reconstitution technology (INSYRT), the scientists use acyl maltose thioesters to extract GPCRs from cells, forming micelles containing the proteins. They t...

Application of Reconstitution Technique on Performance Evaluation of Anchor Bolts

The impact property of a high-strength grade 42CrMoE alloy steel was investigated under varied insert lengths using reconstitution method. A new full-field strain measurement method was employed to explore the plastic deformation region of the steel. Meanwhile, in order to quantitatively examine the insert length influence, an instrumented impact method was proposed to analysis the impact property. The results have successfully demonstrated that inserts equal to or larger than 20 mm can be used to obtain valid data for 42CrMoE.

Membrane Lipid Composition Regulates Alpha-Synuclein Blockage of and Translocation through the Mitochondrial Voltage-Dependent Anion Channel

Alpha-synuclein (α-syn), an intrinsically disordered protein, is a major component of Lewy bodies, a hallmark of Parkinson's disease (PD). α-Syn has been found to interact with both inner and outer mitochondrial membranes (MOM), impair electron transport complexes, and lead to neuronal apoptosis. Recently, using a channel reconstitution technique, we demonstrated that monomeric α-syn both reversibly blocks and translocates through the voltage-dependent anion channel (VDAC) at nanomolar concentrations. Considering VDAC's major role in regulating metabolite fluxes across the MOM, a functional interaction between α-syn and VDAC could be essential for physiological adaptation of mitochondria and dysfunction in PD. Here we show that the α-syn-VDAC interaction is modulated by membrane lipid composition relevant to MOM. We have found that the on-rate of VDAC blockage by α-syn increases up to 10-fold with increase of phosphoethanolamine (PE) content in phosphocholine (PC) membranes. Remarkably, the off-rate was also lipid-dependent, as seen by a 5 mV increase of the turnover potential separating regimes of blockage and translocation with the increase of PE content. We have also found that at physiologically low salt concentrations, the on-rates increase more than 10-fold compared to experiments performed in high salt, while translocation of α-syn through VDAC is impeded. α-Syn differential binding to lipid membranes was also tested using independent methods. These results suggest that the blockage of VDAC by α-syn involves α-syn interaction with the membrane and is governed by both hydrophobic and electrostatic components. Such evidence provides further support for our hypothesis that α-syn needs to bind to the membrane prior to blocking and translocating through the VDAC pore. We propose a new regulatory role of mitochondrial lipids in the (patho-)physiology of monomeric α-syn interaction with mitochondria.

Monitoring of RNA Dynamics in Living Cells Using PUM-HD and Fluorescent Protein Reconstitution Technique.

Fluorescence live-cell RNA imaging to monitor the intracellular localization and dynamics of the target RNA is a challenging subject. One of the difficulties to achieve this is to establish a precise method to enable a fluorescent labeling to the target RNA in living cells. Technologies to reduce the background fluorescence and to detect the RNA with high sensitivity are also necessary to visualize and analyze the intracellular localization and dynamic of the target RNA precisely. Especially in monitoring single-molecule motion, a special setup of a microscope system is required. Such technical problems make the live-cell RNA imaging to be a difficult subject. We recently developed a methodology to label and to visualize a target RNA in living cells with low background fluorescence by using a probe that is based on an RNA-binding protein domain PUM-HD (pumilio homology domain) and a fluorescent protein reconstitution method. A noteworthy property of PUM-HD to apply RNA probes is that this protein domain can be modified to recognize a particular 8-base RNA sequence by inducing tailor-made designed mutagenesis. The fluorescent protein reconstitution method allows us to detect the target RNA with high signal-to-noise ratio. Using the probe based on PUM-HD, a fluorescent protein reconstitution method, and a homebuilt fluorescent microscope system, we succeeded in single-molecule observation of a target RNA in living cells. In this chapter, the techniques to establish the probe and to observe the motion of single-molecule RNA are described.