Multienzyme Mimicking Cascade Mn3O4 Catalyst to Augment Reactive Oxygen Species Elimination and Colorimetric Detection: A Study of Phase Variation upon Calcination Temperature.

Over decades, nanozyme has served as a better replacement of bioenzymes and fulfills most of the shortcomings and intrinsic disadvantages of bioenzymes. Recently, manganese-based nanomaterials have been highly noticed for redox-modulated multienzyme mimicking activity and wide applications in biosensing and biomedical science. The redox-modulated multienzyme mimicking activity was highly in tune with their size, surface functionalization, and charge on the surface and phases. On the subject of calcination temperature to Mn3O4 nanoparticles (NPs), its phase has been transformed to Mn2O3 NPs and Mn5O8 NPs upon different calcination temperatures. Assigning precise structure-property connections is made easier by preparing the various manganese oxides in a single step. The present study has focused on the variation of multienzyme mimicking activity with different phases of Mn3O4 NPs, so that they can be equipped for multifunctional activity with greater potential. Herein, spherical Mn3O4 NPs have been synthesized via a one-step coprecipitation method, and other phases are obtained by direct calcination. The calcination temperature varies to 100, 200, 400, and 600 °C and the corresponding manganese oxide NPs are named M-100, M-200, M-400, and M-600, respectively. The phase transformation and crystalline structure are evaluated by powder X-ray diffraction and selected-area electron diffraction analysis. The different surface morphologies are easily navigated by Fourier transform infrared, field-emission scanning electron microscopy, and high-resolution transmission electron microscopy analysis. Fortunately, for the mixed valence state of Mn3O4 NPs, all phases of manganese oxide NPs showed multienzyme mimicking activity including superoxide dismutase (SOD), catalase, oxidase (OD), and peroxidase; therefore, it offers a synergistic antioxidant ability to overexpose reactive oxygen species. Mn3O4 NPs exhibited good SOD-like enzyme activity, which allowed it to effectively remove the active oxygen (O2•-) from cigarette smoke. A sensitive colorimetric sensor with a low detection limit and a promising linear range has been designed to detect two isomeric phenolic pollutants, hydroquinone (H2Q) and catechol (CA), by utilizing optimized OD activity. The current probe has outstanding sensitivity and selectivity as well as the ability to visually detect two isomers with the unaided eye.

A Pyrene Coupled Azaine-linkage Chromo-fluorogenic Probe for Specific Detection of Sarin Gas Stimulant, Diethylchlorophosphate.

Owing to the extreme toxicity and easy synthesis protocol of G-series nerve agents, developing an efficient sensor for selective detection is necessary. Although various traditional methods are utilized to identify these nerve agents, chromo-fluorogenic probes have gained attractive attention from the scientific communities. In the present contribution, we have introduced a new symmetrical aza-substituted chromo-fluorogenic sensor, BPH, for specific detection of sarin gas, one of the fatal G-series nerve agents surrogate, diethylchlorophosphate (DCP). BPH shows a noticeable naked eye colorimetric change from pale yellow to light pink in the presence of DCP, displaying highly intense bright greenish cyan color photoluminosity under a 365 nm UV lamp,which is also manifested from the color chromaticity diagram. A BPH-staining paper stirps-based test kit experiment has been demonstrated for the on-site detection of nerve agent mimics. A more attractive and efficient application of BPH as a sarin gas vapor phase sensor mimics DCP in solid and solution phases. The BPH-based chromo-fluorogenic sensor shows excellent selectivity toward DCP with a detection and quantification limit in the µM range. This report invokes a new way for the researchers to detect DCP employing a simple chromo-fluorogenic sensor, which could be prepared by a time-saving, straightforward, handy protocol from the cost-effective starting materials.

Label-Free Detection of Epinephrine Using Flower-like Biomimetic CuS Antioxidant Nanozymes.

A biosensor comprising crystalline CuS nanoparticles (NPs) was synthesized via a one-step simple coprecipitation route without involvement of a surfactant. The powder X-ray diffraction method has been used to evaluate the crystalline nature and different phases consist of the formation of CuS NPs. Mainly hexagonal unit cells consist of the formation of CuS NP unit cells. Most of the surfaces are covered with rhombohedral microparticles with a smooth exterior and surface clustering, examined by SEM images, and the shape of NPs was spherical, having an average size of 23 nm, as confirmed by TEM analysis. This study has focused on the peroxidase-mimicking activity, superoxide dismutase (SOD)-mimicking activity, and chemosensor-based colorimetric determination and detection of epinephrine (EP) neurotransmitters with excellent selectivity. The CuS NPs catalyzed the oxidation of the oxidase substrate 3, 3-5, 5 tetramethyl benzidine (TMB) with the help of supplementary H2O2 that followed Michaelis-Menten kinetics with excellent Km and Vmax values calculated by the Lineweaver-Burk plot. Taking advantage of the drop in absorbance upon introduction of EP for the CuS NPs-TMB/H2O2 system, a colorimetric route has been developed for selective and real-time detection of EP. The sensitivity of the new colorimetric probe was vibrant, having a linear range of 0-16 µM, and achieved a low limit of detection of 457 nM. Moreover, the present nanosystem exhibited appreciable SOD-mimicking activity which could effectively remove O2•- from commercial cigarette smoke, along with it acting as a potential radical scavenger as well. The new nanosystem effectively scavenged •OH, O2.-, and metal chelation which were investigated calorimetrically.

Fe-Mn nanocomposites doped graphene quantum dots alleviate salt stress of Triticum aestivum through osmolyte accumulation and antioxidant defense.

An investigation was carried out to evaluate the effect of graphene quantum dots (GQD) and its nanocomposites on germination, growth, biochemical, histological, and major ROS detoxifying antioxidant enzyme activities involved in salinity stress tolerance of wheat. Seedlings were grown on nutrient-free sand and treatment solutions were applied through solid matrix priming and by foliar spray. Control seedlings under salinity stress exhibited a reduction in photosynthetic pigment, sugar content, growth, increased electrolyte leakage, and lipid peroxidation, whereas iron-manganese nanocomposites doped GQD (FM_GQD) treated seedlings were well adapted and performed better compared to control. Enzymatic antioxidants like catalase, peroxidase, glutathione reductase and NADPH oxidase were noted to increase by 40.5, 103.2, 130.19, and 141.23% respectively by application of FM_GQD. Histological evidence confirmed a lower extent of lipid peroxidation and safeguarding the plasma membrane integrity through osmolyte accumulation and redox homeostasis. All of these interactive phenomena lead to an increment in wheat seedling growth by 28.06% through FM_GQD application. These findings highlight that micronutrient like iron, manganese doped GQD can be a promising nano-fertilizer for plant growth and this article will serve as a reference as it is the very first report regarding the ameliorative role of GQD in salt stress mitigation.

Exploration of Diverse Interactions of l-Methionine in Aqueous Ionic Liquid Solutions: Insights from Experimental and Theoretical Studies.

Here, we have investigated some physicochemical parameters to understand the molecular interactions by means of density (ρ) measurement, measurement of viscosity (η), refractive index(n D) measurement, and conductance and surface tension measurements between two significant aqueous ionic liquid solutions: benzyl trimethyl ammonium chloride (BTMAC) and benzyl triethyl ammonium chloride (BTEAC) in an aqueous l-methionine (amino acid) solution. The apparent molar volume (Φv), coefficient of viscosity (B), and molar refraction (R M) have been used to analyze the molecular interaction behavior associated in the solution at various concentrations and various temperatures. With the help of some important equations such as the Masson equation, the Jones-Doles equation, and the Lorentz-Lorenz equation, very significant parameters, namely, limiting apparent molar volumes (Φv 0 ), coefficient of viscosity (B), and limiting molar refraction (R M 0), respectively, are obtained. These parameters along with specific conductance (κ) and surface tension (σ) are very much helpful to reveal the solute-solvent interactions by varying the concentration of solute molecules and temperature in the solution. Analyses of Δµ1 0#, Δµ2 0#, TΔS 2 0#, ΔH 2 0#, and thermodynamic data provide us valuable information about the interactions. We note that l-Met in 0.005 molality BTEAC ionic liquid at 308.15 K shows maximum solute-solvent interaction, while l-Met in 0.001 molality BTMAC aqueous solution of ionic liquid at 298.15 K shows the minimum one. Spectroscopic techniques such as Fourier transform infrared (FTIR), 1H-NMR, and UV-vis also provide supportive information about the interactions between the ionic liquid and l-methionine in aqueous medium. Furthermore, adsorption energy, reduced density gradient (RDG), and molecular electrostatic potential (MESP) maps obtained by the application of density functional theory (DFT) have been used to determine the type of interactions, which are concordant with the experimental observations.

Host-Guest Encapsulation of RIBO with TSC4X: Synthesis, Characterization, and Its Application by Physicochemical and Computational Investigations.

In our present work, we synthesized a new encapsulated complex denoted as RIBO-TSC4X, which was derived from an important vitamin riboflavin (RIBO) and p-sulfonatothiacalix[4]arene(TSC4X). The synthesized complex RIBO-TSC4X was then characterized by utilizing several spectroscopic techniques such as 1H-NMR, FT-IR, PXRD, SEM, and TGA. Job's plot has been employed to show the encapsulation of RIBO (guest) with TSC4X (host) having a 1:1 molar ratio. The molecular association constant of the complex entity (RIBO-TSC4X) was found to be 3116.29 ± 0.17 M-1, suggesting the formation of a stable complex. The augment in aqueous solubility of the RIBO-TSC4X complex compared to pure RIBO was investigated by UV-vis spectroscopy, and it was viewed that the newly synthesized complex has almost 30 times enhanced solubility over pure RIBO. The enhancement of thermal stability upto 440 °C for the RIBO-TSC4X complex was examined by TG analysis. This research also forecasts RIBO's release behavior in the presence of CT-DNA, and at the same time, BSA binding study was also carried out. The synthesized RIBO-TSC4X complex exhibited comparatively better free radical scavenging activity, thereby minimizing oxidative injury of the cell as evident from a series of antioxidant and anti-lipid peroxidation assay. Furthermore, the RIBO-TSC4X complex showed peroxidase-like biomimetic activity, which is very useful for several enzyme catalyst reactions.

Assembled Bisphenol A with cyclic oligosaccharide as the controlled release complex to reduce risky effects.

Herein, in order to improve the bioavailability of a non-biodegradable pollutant, inclusion complexation procedures had been used to develop better formulations of this pollutant, Bisphenol A (BPA). In our research, an inclusion complex (IC) of ß-cyclodextrin (ß-CD) with BPA was formed to investigate the effect of ß-CD on the water solubility, anti-oxidant, anti-bacterial activity, toxicity, and thermal stability of BPA. UV-Vis and other spectrometric methods such as NMR, FTIR, and XRD indicated the molecular mechanism of interactions between ß-CD and BPA, which was further hypothesized using molecular modeling to confirm preliminary results. Studies of TGA and DSC demonstrated that encapsulation boosted the thermal stability of BPA. This research also makes predictions about BPA's release behavior when CT-DNA is present. In vitro testing of the IC's antibacterial activities showed that it outperformed pure BPA. The in silico study was found to have a considerable decrease in toxicity level for IC compared to pure BPA. Therefore, ß-CD-encapsulated BPA can lessen toxicity by raising antioxidant levels. Additionally, as its antibacterial activity increases, it may be employed therapeutically. Thus, this discovery of creating BPA formulations with controlled release and/or protective properties allows for a more logical application of BPA by reducing its hazardous effects through boosting its efficacy.

Exploring inclusion complex of an anti-cancer drug (6-MP) with ß-cyclodextrin and its binding with CT-DNA for innovative applications in anti-bacterial activity and photostability optimized by computational study.

The co-evaporation approach was used to examine the host-guest interaction and to explore the cytotoxic and antibacterial properties of an important anti-cancer medication, 6-mercaptopurine monohydrate (6-MP) with ß-cyclodextrin (ß-CD). The UV-Vis investigation confirmed the inclusion complex's (IC) 1 : 1 stoichiometry and was also utilized to oversee the viability of this inclusion process. FTIR, NMR, and XRD, among other spectrometric techniques, revealed the mechanism of molecular interactions between ß-CD and 6-MP which was further hypothesized by DFT to verify tentative outcomes. TGA and DSC studies revealed that 6-MP's thermal stability increased after encapsulation. Because of the protection of drug 6-MP by ß-CD, the formed IC was found to have higher photostability. This work also predicts the release behavior of 6-MP in the presence of CT-DNA without any chemical changes. An evaluation of the complex's antibacterial activity in vitro revealed that it was more effective than pure 6-MP. The in vitro cytotoxic activity against the human kidney cancer cell line (ACHN) was also found to be significant for the IC (IC50 = 4.18 µM) compared to that of pure 6-MP (IC50 = 5.49 µM). These findings suggest that 6-MP incorporation via ß-CD may result in 6-MP stability and effective presentation of its solubility, cytotoxic and antibacterial properties.

Intrinsic Light-Activated Oxidase Mimicking Activity of Conductive Polyaniline Nanofibers: A Class of Metal-Free Nanozyme.

In recent decades, studies have focused on inorganic nanozymes to overcome the intrinsic drawbacks of bioenzymes due to the demands of improving the reaction conditions and lack of robustness to harsh environmental factors. Many biochemical reactions catalyzed by enzymes require light activation. Light-activated nanozymes have distinct advantages, including being regulated by light stimuli, activating the molecular oxygen to produce reactive oxygen species (ROS) without interfering supplementary oxidants, and often showing a synergistic effect to catalyze some challenging reactions. Only a few studies have been done on this connection. Therefore, it is still a big challenge to develop a nanozyme regulated by light activation. Herein, we uncovered the light-activated oxidase mimicking activity of a conducting polymer polyaniline nanofibers (PANI-NFs). PANI-NFs exhibit intrinsic light-activated brilliant oxidase-like activity, can catalyze the colorless tetramethyl benzidine (TMB) to produce a blue product TMBox, and have a distinct Km = 0.087 mM and a high Vmax = 2.32 µM min-1 value, measured by using Hanes-Woolf kinetics. We also report the light-activated oxidase activity of some other renowned carbocatalysts graphene oxide and graphitic carbon nitride and compare them with PANI-NFs. This type of property shown by the conductive polymer is amazing. The density functional theory is used to verify the stability and the mode of adsorption of the PANI NFs-TMB composite, which corroborates the experimental results. Furthermore, the current nanozyme demonstrated a significant ability to kill both Gram-negative and Gram-positive bacteria as well as effectively destroy biofilms under physiological conditions. We believe that this work provides the motivation to create a link between optoelectronics and biological activity in the near future.

Probing the Molecular Assembly of a Metabolizer Drug with ß-Cyclodextrin and Its Binding with CT-DNA in Augmenting Antibacterial Activity and Photostability by Physicochemical and Computational Methodologies.

The assembly of an inclusion complex in an aqueous medium using a metabolizer drug (dyphylline) as guest and ß-cyclodextrin as host has been established, which is extremely appropriate for a variety of applications in modern biomedical sciences. The formation of the inclusion complex is established by 1H NMR, and surface tension and conductivity measurements demonstrate that the inclusion complex was produced with 1:1 stoichiometry. The thermodynamic parameters based on density, viscosity, and refractive index measurements were used to determine the nature of the complex. This research also forecasts how dyphylline will release in the presence of CT-DNA without any chemical modifications. The produced insertion complex (IC) has a higher photostability due to the drug dyphylline being protected by ß-CD. The antibacterial activity of dyphylline greatly improved after complexation and exhibited higher toxicity against Gram-negative (highest against Escherichia coli) in comparison to Gram-positive bacteria. The encapsulation mode of the dyphylline molecule into the cavity of the ß-CD was also investigated using DFT to confirm preliminary results.

Evidence for low-level translation in human erythrocytes.

It is generally believed that human mature erythrocytes do not possess functional ribosomes and therefore cannot synthesize proteins. However, the absence of translation is not consistent with the long lifespan of mature erythrocytes. They stay viable and functional for about 115 d in the circulatory system. Here, using a highly pure preparation of human mature erythrocytes, we demonstrate the presence of translation by polysome profiling, [35S]methionine labeling, and RiboPuromycylation. [35S]methionine labeling revealed that the translation in mature erythrocytes is about 10% of that observed in reticulocytes. We could observe polysomes by transmission electron microscopy in these cells. RNA-seq and quantitative real-time PCR performed on polysome fractions of these cells revealed that HBA (α-globin) and HBB (ß-globin) transcripts are translated. Using a luciferase-based reporter assay and mutational studies, we show that the sequence of the 5' untranslated region is crucial for the translation of these transcripts. Furthermore, mature erythrocytes showed reduced expression of globin proteins (α- and ß-) when treated with translation inhibitors. Overall, we provide multiple lines of evidence for translation of globin mRNAs in human mature erythrocytes.

Low complexity RGG-motif containing proteins Scd6 and Psp2 act as suppressors of clathrin heavy chain deficiency.

Clathrin, made up of the heavy- and light-chains, constitutes one of the most abundant proteins involved in intracellular protein trafficking and endocytosis. YPR129W, which encodes RGG-motif containing translation repressor was identified as a part of the multi-gene construct (SCD6) that suppressed clathrin deficiency. However, the contribution of YPR129W alone in suppressing clathrin deficiency has not been documented. This study identifies YPR129W as a necessary and sufficient gene in a multi-gene construct SCD6 that suppresses clathrin deficiency. Importantly, we also identify cytoplasmic RGG-motif protein encoding gene PSP2 as another novel suppressor of clathrin deficiency. Detailed domain analysis of the two suppressors reveals that the RGG-motif of both Scd6 and Psp2 is important for suppressing clathrin deficiency. Interestingly, the endocytosis function of clathrin heavy chain assayed by internalization of GFP-Snc1 and α-factor secretion activity are not complemented by either Scd6 or Psp2. We further observe that inhibition of TORC1 compromises the suppression activity of both SCD6 and PSP2 to different extent, suggesting that two suppressors are differentially regulated. Scd6 granules increased based on its RGG-motif upon Chc1 depletion. Strikingly, Psp2 overexpression increased the abundance of ubiquitin-conjugated proteins in Chc1 depleted cells in its RGG-motif dependent manner and also decreased the accumulation of GFP-Atg8 foci. Overall based on our results using SCD6 and PSP2, we identify a novel role of RGG-motif containing proteins in suppressing clathrin deficiency. Since both the suppressors are RNA-binding proteins, this study opens an exciting avenue for exploring the connection between clathrin function and post-transcriptional gene control processes.

ß-Cyclodextrin-Stabilized Biosynthesis Nanozyme for Dual Enzyme Mimicking and Fenton Reaction with a High Potential Anticancer Agent.

The myth of inactivity of inorganic materials in a biological system breaks down by the discovery of nanozymes. From this time, the nanozyme has attracted huge attention for its high durability, cost-effective production, and easy storage over the natural enzyme. Moreover, the multienzyme-mimicking activity of nanozymes can regulate the level of reactive oxygen species (ROS) in an intercellular system. ROS can be generated by peroxidase (POD), oxidase (OD), and Fenton-like catalytic reaction by a nanozyme which kills the cancer cells by oxidative stress; therefore, it is important in CDT (chemo dynamic therapy). Our current study designed to investigate the enzyme mimicking behavior and anticancer ability of cerium-based nanomaterials because the cerium-based materials offer a high redox ability while maintaining nontoxicity and high stability. Our group synthesized CeZrO4 nanoparticles by a green method using ß-cyclodextrin as a stabilizer and neem leaf extract as a reducing agent, exhibiting POD- and OD-like dual enzyme activities. The best enzyme catalytic activity is shown in pH = 4, indicating the high ROS generation in an acidic medium (tumor microenvironment) which is also supported by the Fenton-like behavior of CeZrO4 nanoparticles. Inspired by the high ROS generation in vitro method, we investigated the disruption of human kidney cells by this nanoparticle, successfully verified by the MTT assay. The harmful effect of ROS in a normal cell is also investigated by the in vitro MTT assay. The results suggested that the appreciable anticancer activity with minimal side effects by this synthesized nanomaterial.

Encapsulated hydroxychloroquine and chloroquine into cyclic oligosaccharides are the potential therapeutics for COVID-19: insights from first-principles calculations.

Novel-Coronavirus (COVID-19) outburst has become a worldwide pandemic which threaten the scientific community to design and discover efficient and effective treatment strategies against this deadly virus (SARS-CoV-2). Still now, there is no antiviral therapy or drug available in the market which can efficiently combat the infection caused by this virus. In this respect, using available drugs by screening with molecular docking and molecular dynamics studies not only minimizes lengthy chemical trials but also reduces discovery cost for the pharmaceutical industry. During the COVID-19 pandemic situations hydroxychloroquine, chloroquine known as HCQ and CQ tablets have gained popularity as for the treatment coronavirus (COVID-19) but the main threatening effect of HCQ, CQ use lies on their side effects like blistering, peeling, loosening of the skin, blurred vision stomach pain, diarrhea, chest discomfort, pain, or tightness, cough or hoarseness which require immediate medical attention. Encapsulation of HCQ and CQ drugs by the cyclic macromolecules such as α and ß-Cyclodextrin, to form host-guest complexes is very effective strategy to mask the cytotoxicity of certain drugs and alleviating and modulating side effects of drug applications. In the present work, we have encapsulated the HCQ and CQ drugs α and ß-Cyclodextrin and made a comprehensive analysis of stability, optical properties. Details analysis verified that between QC and HCQ, HQC showed stronger affinity towards ß-Cyclodextrin. This strategy can reduce the side effect of HCQ and CQ thereby offers a new way to use these drugs. We hope the present study should help the researchers to develop potential therapeutics against the novel coronavirus.

Exploring the Inclusion Complex of a Drug (Umbelliferone) with α-Cyclodextrin Optimized by Molecular Docking and Increasing Bioavailability with Minimizing the Doses in Human Body.

In this study, umbelliferone and α-cyclodextrin host molecules have been mixed up through a coprecipitation method to prepare a supramolecular complex to provide physical insights into the formation and stability of the inclusion complex (IC). The prepared hybrid was characterized by 1H nuclear magnetic resonance (1H NMR), Fourier transform infrared (FTIR) spectroscopy, electrospray ionization (ESI) mass spectrometry, DSC, and fluorescence spectroscopic studies. Job's plot provides a stoichiometric ratio of 1:1 and the Benesi-Hildebrand double reciprocal plot gives binding constant values using fluorescence spectroscopic titrations and the ESI mass data support the experimental observations. The results of molecular modeling were systematically analyzed to validate the inclusion complexation. In preliminary computational screening, α-cyclodextrin IC of umbelliferone was found to be quite stable based on the docking score, binding free energies, and dynamic simulations. In addition, the results obtained from 1H NMR and FTIR spectroscopy studies supported the inclusion complexation phenomenon. The results obtained from computational studies were found to be consistent with the experimental data to ascertain the encapsulation of umbelliferone into α-cyclodextrin.

Integrated Regulation of HuR by Translation Repression and Protein Degradation Determines Pulsatile Expression of p53 Under DNA Damage.

Expression of tumor suppressor p53 is regulated at multiple levels, disruption of which often leads to cancer. We have adopted an approach combining computational systems modeling with experimental validation to elucidate the translation regulatory network that controls p53 expression post DNA damage. The RNA-binding protein HuR activates p53 mRNA translation in response to UVC-induced DNA damage in breast carcinoma cells. p53 and HuR levels show pulsatile change post UV irradiation. The computed model fitted with the observed pulse of p53 and HuR only when hypothetical regulators of synthesis and degradation of HuR were incorporated. miR-125b, a UV-responsive microRNA, was found to represses the translation of HuR mRNA. Furthermore, UV irradiation triggered proteasomal degradation of HuR mediated by an E3-ubiquitin ligase tripartite motif-containing 21 (TRIM21). The integrated action of miR-125b and TRIM21 constitutes an intricate control system that regulates pulsatile expression of HuR and p53 and determines cell viability in response to DNA damage.

Suppressor of clathrin deficiency (Scd6)-An emerging RGG-motif translation repressor.

Translation control plays a key role in variety of cellular processes. Translation initiation factors augment translation, whereas translation repressor proteins inhibit translation. Different repressors act by distinct mechanisms to accomplish the repression process. Although messenger RNAs (mRNAs) can be repressed at various steps of translation, most repressors have been reported to target the initiation step. We focus on one such translation repressor, an Arginine-Glycine-Glycine (RGG)-motif containing protein Scd6. Using this protein as a model, we present a discourse on the known and possible functions of this repressor, its mechanism of action and its recently reported regulation. We suggest a case for conservation of the mechanism employed by Scd6 along with its regulation in orthologs, and propose that Scd6 family of proteins will be an ideal tool to understand translation control and mRNA fate decision mechanisms across biological systems. This article is categorized under: Translation > Translation Regulation RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.