ResearchPad - monomers https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Atco, a yeast mitochondrial complex of Atp9 and Cox6, is an assembly intermediate of the ATP synthase]]> https://www.researchpad.co/article/elastic_article_14724 Mitochondrial oxidative phosphorylation (oxphos) is the process by which the ATP synthase conserves the energy released during the oxidation of different nutrients as ATP. The yeast ATP synthase consists of three assembly modules, one of which is a ring consisting of 10 copies of the Atp9 subunit. We previously reported the existence in yeast mitochondria of high molecular weight complexes composed of mitochondrially encoded Atp9 and of Cox6, an imported structural subunit of cytochrome oxidase (COX). Pulse-chase experiments indicated a correlation between the loss of newly translated Atp9 complexed to Cox6 and an increase of newly formed Atp9 ring, but did not exclude the possibility of an alternate source of Atp9 for ring formation. Here we have extended studies on the functions and structure of this complex, referred to as Atco. We show that Atco is the exclusive source of Atp9 for the ATP synthase assembly. Pulse-chase experiments show that newly translated Atp9, present in Atco, is converted to a ring, which is incorporated into the ATP synthase with kinetics characteristic of a precursor-product relationship. Even though Atco does not contain the ring form of Atp9, cross-linking experiments indicate that it is oligomeric and that the inter-subunit interactions are similar to those of the bona fide ring. We propose that, by providing Atp9 for biogenesis of ATP synthase, Atco complexes free Cox6 for assembly of COX. This suggests that Atco complexes may play a role in coordinating assembly and maintaining proper stoichiometry of the two oxphos enzymes

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<![CDATA[Amino acids serve as an important energy source for adult flukes of <i>Clonorchis sinensis</i>]]> https://www.researchpad.co/article/elastic_article_13829 Clonorchiasis, closely related to cholangiocarcinoma and hepatocellular carcinoma, has led to a negative socioeconomic impact in global areas especially some Asian endemic regions. Owing to the emergence of drug resistance and hypersensitivity reactions after the massive and repeated use of praziquantel as well as the lack of effective vaccines, searching for new strategies that prevent and treat clonorchiasis has become an urgent matter. Clonorchis sinensis, the causative agent of clonorchiasis, long-term inhabits the microaerobic and limited-glucose environment of the bile ducts. Adequate nutrients are essential for adult flukes to resist the adverse condition and survive in the crowed habitat. Studies on energy metabolism of adult flukes are beneficial for further exploring host-parasite interactions and developing novel anti-parasitic drugs. Our results suggest that gluconeogenesis probably plays a vital role in energy metabolism of Clonorchis sinensis and exogenous amino acids might be an essential energy source for adult flukes to successfully survive in the host. Our foundational study opens a new avenue for explaining energy metabolism of Clonorchis sinensis and provides a valuable strategy that the gluconeogenesis pathway will be a potential and novel target for the prevention and treatment of clonorchiasis.

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<![CDATA[Crystal structures of Triosephosphate Isomerases from Taenia solium and Schistosoma mansoni provide insights for vaccine rationale and drug design against helminth parasites]]> https://www.researchpad.co/article/N340e3046-cb91-4c84-8d1b-fb2a65cf4cdb

Triosephosphate isomerases (TPIs) from Taenia solium (TsTPI) and Schistosoma mansoni (SmTPI) are potential vaccine and drug targets against cysticercosis and schistosomiasis, respectively. This is due to the dependence of parasitic helminths on glycolysis and because those proteins elicit an immune response, presumably due to their surface localization. Here we report the crystal structures of TsTPI and SmTPI in complex with 2-phosphoglyceric acid (2-PGA). Both TPIs fold into a dimeric (β-α)8 barrel in which the dimer interface consists of α-helices 2, 3, and 4, and swapping of loop 3. TPIs from parasitic helminths harbor a region of three amino acids knows as the SXD/E insert (S155 to E157 and S157 to D159 in TsTPI and SmTPI, respectively). This insert is located between α5 and β6 and is proposed to be the main TPI epitope. This region is part of a solvent-exposed 310–helix that folds into a hook-like structure. The crystal structures of TsTPI and SmTPI predicted conformational epitopes that could be used for vaccine design. Surprisingly, the epitopes corresponding to the SXD/E inserts are not the ones with the greatest immunological potential. SmTPI, but not TsTPI, habors a sole solvent exposed cysteine (SmTPI-S230) and alterations in this residue decrease catalysis. The latter suggests that thiol-conjugating agents could be used to target SmTPI. In sum, the crystal structures of SmTPI and TsTPI are a blueprint for targeted schistosomiasis and cysticercosis drug and vaccine development.

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<![CDATA[Water-induced strong protection against acute exposure to low subzero temperature of adult Aedes albopictus]]> https://www.researchpad.co/article/5c61e909d5eed0c48496f6ed

As an important vector of dengue and Zika, Aedes albopictus has been the fastest spreading invasive mosquitoes in the world over the last 3–4 decades. Cold tolerance is important for survival and expansion of insects. Ae. albopictus adults are generally considered to be cold-intolerant that cannot survive at subzero temperature. However, we found that Ae. albopictus could survive for several hours’ exposure to -9 to -19 oC so long as it was exposed with water. Median lethal time (LT50) of Ae. albopictus exposed to -15 and -19 oC with water increased by more than 100 times compared to those exposed to the same subzero temperature without water. This phenomenon also existed in adult Aedes aegypti and Culex quinquefasciatus. Ae. albopictus female adults which exposed to low subzero temperature at -9 oC with water had similar longevity and reproductive capacity to those of females without cold exposure. Cold exposure after a blood meal also have no detrimental impact on survival capacity of female adult Ae. albopictus compared with those cold exposed without a blood meal. Moreover, our results showed that rapid cold hardening (RCH) was induced in Ae. albopictus during exposing to low subzero temperature with water. Both the RCH and the relative high subzero temperature of water immediate after cold exposure might provide this strong protection against low subzero temperature. The molecular basis of water-induced protection for Ae. albopictus might refer to the increased glycerol during cold exposure, as well as the increased glucose and hsp70 during recovery from cold exposure. Our results suggested that the water-induced strong protection against acute decrease of air temperature for adult mosquitoes might be important for the survival and rapid expansion of Ae. albopictus.

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<![CDATA[Aggregation and interfacial phenomenon of amphiphilic drug under the influence of pharmaceutical excipients (green/biocompatible gemini surfactant)]]> https://www.researchpad.co/article/5c648cbcd5eed0c484c81680

In the current study, we have examined the interaction amongst an antidepressant drug amitriptyline hydrochloride (AMH) and ethane-1, 2-diyl bis(N,N-dimethyl-N-cetylammoniumacetoxy) dichloride (16-E2-16, a green gemini surfactant) through tensiometric and fluorimetric techniques in aqueous/electrolyte/urea solutions. Significant variations are observed in the various evaluated parameters in the present study. Gemini 16-E2-16 has outstanding surface properties along with a much lower cmc value, demonstrating very little toxicity as well as considerable antimicrobial activity. The cmc values of mixtures decrease through increase in mole fraction (α1) of 16-E2-16, which specifies the nonideality of the solution mixtures, along with demonstrating the occurrence of mixed micellization too. Negative βRub values signify on the whole attractive force of interaction between constituents of mixed micelles. Owing to the incidence of electrolyte NaCl (50 mmol.kg–1), lowering of the micelles’ surface charge happens, resulting in aggregation taking place at lower concentration while the presence of urea (NH2CONH2) halts micellization taking place, which means the cmc value increases in the attendance of urea. The ΔGmo values for all systems were negative along with the presence of electrolyte/urea. The excess free energy (Gex) of studied mixed systems was also estimated and found to be negative for all the systems. Using the fluorescence quenching method, the micelle aggregation number (Nagg) was evaluated and it was found that the contribution of gemini surfactant was always more than that of the AMH and their value enhances in the existence of electrolyte while decreasing in the attendance of NH2CONH2 in the system. In addition, other fluorescence parameters such as micropolarity (I1/I3), dielectric constant (Dexp) as well as Stern–Volmer binding constants (Ksv) of mixed systems were evaluated and the results showed the synergistic performance of the AMH + 16-E2-16 mixtures. Along with tensiometric and fluorimetric techniques, FT-IR spectroscopy was also engaged to reveal the interaction among constituents.

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<![CDATA[R pyocin tail fiber structure reveals a receptor-binding domain with a lectin fold]]> https://www.researchpad.co/article/5c633948d5eed0c484ae63f5

R pyocins are ɸCTX-like myophage tailocins of Pseudomonas sp. Adsorption of R pyocins to target strains occurs by the interaction of tail fiber proteins with core lipopolysaccharide (LPS). Here, we demonstrate that N-terminally truncated R pyocin tail fibers corresponding to a region of variation between R-subtypes are sufficient to bind target strains according to R-subtype. We also report the crystal structures of these tail fiber proteins and show that they form an elongated helical trimer composed of three domains arranged linearly from N- to C-terminus: a baseplate proximal head, medial shaft, and distal foot. The head and shaft domains contain novel structural motifs. The foot domain, however, is composed of a conserved jellyroll fold and shares high structural similarity to the tail fiber of myophage AP22, podophage tailspike C-terminal domains (LKA-1 and ɸ297), and several eukaryotic adhesins (discoidin I/II, agglutinin, and octocoral lectin). Many of these proteins bind polysaccharides by means of their distal loop network, a series of highly variable loops at one end of the conserved jellyroll fold backbone. Our structures reveal that the majority of R-subtype specific polymorphisms cluster in patches covering a cleft formed at the oligomeric interface of the head domain and in a large patch covering much of the foot domain, including the distal loop network. Based on the structural variation in distal loops within the foot region, we propose that the foot is the primary sugar-binding domain of R pyocins and R-subtype specific structural differences in the foot domain distal loop network are responsible for binding target strains in an R-subtype dependent manner.

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<![CDATA[Structural basis of DSF recognition by its receptor RpfR and its regulatory interaction with the DSF synthase RpfF]]> https://www.researchpad.co/article/5c61e8f4d5eed0c48496f4de

The diffusible signal factors (DSFs) are a family of quorum-sensing autoinducers (AIs) produced and detected by numerous gram-negative bacteria. The DSF family AIs are fatty acids, differing in their acyl chain length, branching, and substitution but having in common a cis-2 double bond that is required for their activity. In both human and plant pathogens, DSFs regulate diverse phenotypes, including virulence factor expression, antibiotic resistance, and biofilm dispersal. Despite their widespread relevance to both human health and agriculture, the molecular basis of DSF recognition by their cellular receptors remained a mystery. Here, we report the first structure–function studies of the DSF receptor regulation of pathogenicity factor R (RpfR). We present the X-ray crystal structure of the RpfR DSF-binding domain in complex with the Burkholderia DSF (BDSF), which to our knowledge is the first structure of a DSF receptor in complex with its AI. To begin to understand the mechanistic role of the BDSF–RpfR contacts observed in the biologically important complex, we have also determined the X-ray crystal structure of the RpfR DSF-binding domain in complex with the inactive, saturated isomer of BDSF, dodecanoic acid (C12:0). In addition to these ligand–receptor complex structures, we report the discovery of a previously overlooked RpfR domain and show that it binds to and negatively regulates the DSF synthase regulation of pathogenicity factor F (RpfF). We have named this RpfR region the RpfF interaction (FI) domain, and we have determined its X-ray crystal structure alone and in complex with RpfF. These X-ray crystal structures, together with extensive complementary in vivo and in vitro functional studies, reveal the molecular basis of DSF recognition and the importance of the cis-2 double bond to DSF function. Finally, we show that throughout cellular growth, the production of BDSF by RpfF is post-translationally controlled by the RpfR N-terminal FI domain, affecting the cellular concentration of the bacterial second messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). Thus, in addition to describing the molecular basis for the binding and specificity of a DSF for its receptor, we describe a receptor–synthase interaction regulating bacterial quorum-sensing signaling and second messenger signal transduction.

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<![CDATA[Protein refolding based on high hydrostatic pressure and alkaline pH: Application on a recombinant dengue virus NS1 protein]]> https://www.researchpad.co/article/5c6448dbd5eed0c484c2f04f

In this study we evaluated the association of high hydrostatic pressure (HHP) and alkaline pH as a minimally denaturing condition for the solubilization of inclusion bodies (IBs) generated by recombinant proteins expressed by Escherichia coli strains. The method was successfully applied to a recombinant form of the dengue virus (DENV) non-structural protein 1 (NS1). The minimal pH for IBs solubilization at 1 bar was 12 while a pH of 10 was sufficient for solubilization at HHP: 2.4 kbar for 90 min and 0.4 kbar for 14 h 30 min. An optimal refolding condition was achieved by compression of IBs at HHP and pH 10.5 in the presence of arginine, oxidized and reduced glutathiones, providing much higher yields (up to 8-fold) than association of HHP and GdnHCl via an established protocol. The refolded NS1, 109 ± 9.5 mg/L bacterial culture was recovered mainly as monomer and dimer, corresponding up to 90% of the total protein and remaining immunologically active. The proposed conditions represent an alternative for the refolding of immunologically active recombinant proteins expressed as IBs.

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<![CDATA[Novel anti-biofouling bioactive calcium silicate-based cement containing 2-methacryloyloxyethyl phosphorylcholine]]> https://www.researchpad.co/article/5c61b7ded5eed0c4849380ad

Calcium silicate-based cements (CSCs) are commonly used for endodontic procedures; however, their antibacterial effects are limited. The objective of this study was to develop a 2-methacryloyloxyethyl phosphorylcholine (MPC)-incorporated CSC with improved antibacterial properties, while maintaining the original advantageous features of CSC. MPC was incorporated into a commercial CSC (Endocem MTA) at 0 wt% (control), 1.5%, 3.0 wt%, 5.0 wt%, 7.5 wt%, and 10 wt%. The setting time, compressive strength, water sorption, and glycerol contact angle were measured. Protein absorption was measured and bacterial adhesion on the surface was evaluated using Enterococcus faecalis. The bactericidal effect was examined by the disc diffusion test. Mineralization ability was assessed based on calcium ion deposition, as assessed by alizarin red staining, after immersion into Hank’s balanced salt solution for 7 days. High concentrations of MPC in CSC (7.5 wt% and 10 wt%) increased the setting time, reduced compressive strength, and reduced wettability. MPC (3 wt%) had greater protein repellent and anti-biofouling effects than those of control and test materials (P < 0.001). However, no bactericidal effect was observed for any control or test materials. There was greater calcium ion deposition on the surface of MPC-supplemented CSC than on the control (P < 0.001). The addition of 3 wt% MPC polymer to CSC confers protein-repellent properties and reduced bacterial attachment, with the potential for improved mineralization.

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<![CDATA[Hydroxycholesterol binds and enhances the anti-viral activities of zebrafish monomeric c-reactive protein isoforms]]> https://www.researchpad.co/article/5c61b7d0d5eed0c484937fef

C-reactive proteins (CRPs) are among the faster acute-phase inflammation-responses proteins encoded by one gene (hcrp) in humans and seven genes (crp1-7) in zebrafish (Danio rerio) with importance in bacterial and viral infections. In this study, we described novel preferential bindings of 25-hydroxycholesterol (25HOCh) to CRP1-7 compared with other lipids and explored the antiviral effects of both 25HOCh and CRP1-7 against spring viremia carp virus (SVCV) infection in zebrafish. Both in silico and in vitro results confirmed the antiviral effect of 25HOCh and CRP1-7 interactions, thereby showing that the crosstalk between them differed among the zebrafish isoforms. The presence of oxidized cholesterols in human atherosclerotic plaques amplifies the importance that similar interactions may occur for vascular and/or neurodegenerative diseases during viral infections. In this context, the zebrafish model offers a genetic tool to further investigate these interactions.

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<![CDATA[Advancing multimer analysis of von Willebrand factor by single-molecule AFM imaging]]> https://www.researchpad.co/article/5c478c8ed5eed0c484bd3141

The formation of hemostatic plugs at sites of vascular injury crucially involves the multimeric glycoprotein von Willebrand factor (VWF). VWF multimers are linear chains of N-terminally linked dimers. The latter are formed from monomers via formation of the C-terminal disulfide bonds Cys2771-Cys2773’, Cys2773-Cys2771’, and Cys2811-Cys2811’. Mutations in VWF that impair multimerization can lead to subtype 2A of the bleeding disorder von Willebrand Disease (VWD). Commonly, the multimer size distribution of VWF is assessed by electrophoretic multimer analysis. Here, we present atomic force microscopy (AFM) imaging as a method to determine the size distribution of VWF variants by direct visualization at the single-molecule level. We first validated our approach by investigating recombinant wildtype VWF and a previously studied mutant (p.Cys1099Tyr) that impairs N-terminal multimerization. We obtained excellent quantitative agreement with results from earlier studies and with electrophoretic multimer analysis. We then imaged specific mutants that are known to exhibit disturbed C-terminal dimerization. For the mutants p.Cys2771Arg and p.Cys2773Arg, we found the majority of monomers (87 ± 5% and 73 ± 4%, respectively) not to be C-terminally dimerized. While these results confirm that Cys2771 and Cys2773 are crucial for dimerization, they additionally provide quantitative information on the mutants’ different abilities to form alternative C-terminal disulfides for residual dimerization. We further mutated Cys2811 to Ala and found that only 23 ± 3% of monomers are not C-terminally dimerized, indicating that Cys2811 is structurally less important for dimerization. Furthermore, for mutants p.Cys2771Arg, p.Cys2773Arg, and p.Cys2811Ala we found ‘even-numbered’ non-native multimers, i.e. multimers with monomers attached on both termini; a multimer species that cannot be distinguished from native multimers by conventional multimer analysis. Summarizing, we demonstrate that AFM imaging can provide unique insights into VWF processing defects at the single-molecule level that cannot be gained from established methods of multimer analysis.

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<![CDATA[Crystal structure of chloramphenicol-metabolizing enzyme EstDL136 from a metagenome]]> https://www.researchpad.co/article/5c478c8dd5eed0c484bd306a

Metagenomes often convey novel biological activities and therefore have gained considerable attention for use in biotechnological applications. Recently, metagenome-derived EstDL136 was found to possess chloramphenicol (Cm)-metabolizing features. Sequence analysis showed EstDL136 to be a member of the hormone-sensitive lipase (HSL) family with an Asp-His-Ser catalytic triad and a notable substrate specificity. In this study, we determined the crystal structures of EstDL136 and in a complex with Cm. Consistent with the high sequence similarity, the structure of EstDL136 is homologous to that of the HSL family. The active site of EstDL136 is a relatively shallow pocket that could accommodate Cm as a substrate as opposed to the long acyl chain substrates typical of the HSL family. Mutational analyses further suggested that several residues in the vicinity of the active site play roles in the Cm-binding of EstDL136. These results provide structural and functional insights into a metagenome-derived EstDL136.

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<![CDATA[Chemogenomic model identifies synergistic drug combinations robust to the pathogen microenvironment]]> https://www.researchpad.co/article/5c6059c7d5eed0c4847cbe16

Antibiotics need to be effective in diverse environments in vivo. However, the pathogen microenvironment can have a significant impact on antibiotic potency. Further, antibiotics are increasingly used in combinations to combat resistance, yet, the effect of microenvironments on drug-combination efficacy is unknown. To exhaustively explore the impact of diverse microenvironments on drug-combinations, here we develop a computational framework—Metabolism And GENomics-based Tailoring of Antibiotic regimens (MAGENTA). MAGENTA uses chemogenomic profiles of individual drugs and metabolic perturbations to predict synergistic or antagonistic drug-interactions in different microenvironments. We uncovered antibiotic combinations with robust synergy across nine distinct environments against both E. coli and A. baumannii by searching through 2556 drug-combinations of 72 drugs. MAGENTA also accurately predicted the change in efficacy of bacteriostatic and bactericidal drug-combinations during growth in glycerol media, which we confirmed experimentally in both microbes. Our approach identified genes in glycolysis and glyoxylate pathway as top predictors of synergy and antagonism respectively. Our systems approach enables tailoring of antibiotic therapies based on the pathogen microenvironment.

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<![CDATA[Clostridium butyricum population balance model: Predicting dynamic metabolic flux distributions using an objective function related to extracellular glycerol content]]> https://www.researchpad.co/article/5c25450ad5eed0c48442bd72

Background

Extensive experimentation has been conducted to increment 1,3-propanediol (PDO) production using Clostridium butyricum cultures in glycerol, but computational predictions are limited. Previously, we reconstructed the genome-scale metabolic (GSM) model iCbu641, the first such model of a PDO-producing Clostridium strain, which was validated at steady state using flux balance analysis (FBA). However, the prediction ability of FBA is limited for batch and fed-batch cultures, which are the most often employed industrial processes.

Results

We used the iCbu641 GSM model to develop a dynamic flux balance analysis (DFBA) approach to predict the PDO production of the Colombian strain Clostridium sp IBUN 158B. First, we compared the predictions of the dynamic optimization approach (DOA), static optimization approach (SOA), and direct approach (DA). We found no differences between approaches, but the DOA simulation duration was nearly 5000 times that of the SOA and DA simulations. Experimental results at glycerol limitation and glycerol excess allowed for validating dynamic predictions of growth, glycerol consumption, and PDO formation. These results indicated a 4.4% error in PDO prediction and therefore validated the previously proposed objective functions. We performed two global sensitivity analyses, finding that the kinetic input parameters of glycerol uptake flux had the most significant effect on PDO predictions. The other input parameters evaluated during global sensitivity analysis were biomass composition (precursors and macromolecules), death constants, and the kinetic parameters of acetic acid secretion flux. These last input parameters, all obtained from other Clostridium butyricum cultures, were used to develop a population balance model (PBM). Finally, we simulated fed-batch cultures, predicting a final PDO production near to 66 g/L, almost three times the PDO predicted in the best batch culture.

Conclusions

We developed and validated a dynamic approach to predict PDO production using the iCbu641 GSM model and the previously proposed objective functions. This validated approach was used to propose a population model and then an increment in predictions of PDO production through fed-batch cultures. Therefore, this dynamic model could predict different scenarios, including its integration into downstream processes to predict technical-economic feasibilities and reducing the time and costs associated with experimentation.

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<![CDATA[Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes]]> https://www.researchpad.co/article/5c26977bd5eed0c48470fa8f

Intact red blood cells (RBCs) are required for phenotypic analyses. In order to allow separation (time and location) between subject encounter and sample analysis, we developed a research-specific RBC cryopreservation protocol and assessed its impact on data fidelity for key biochemical and physiological assays. RBCs drawn from healthy volunteers were aliquotted for immediate analysis or following glycerol-based cryopreservation, thawing, and deglycerolization. RBC phenotype was assessed by (1) scanning electron microscopy (SEM) imaging and standard morphometric RBC indices, (2) osmotic fragility, (3) deformability, (4) endothelial adhesion, (5) oxygen (O2) affinity, (6) ability to regulate hypoxic vasodilation, (7) nitric oxide (NO) content, (8) metabolomic phenotyping (at steady state, tracing with [1,2,3-13C3]glucose ± oxidative challenge with superoxide thermal source; SOTS-1), as well as in vivo quantification (following human to mouse RBC xenotransfusion) of (9) blood oxygenation content mapping and flow dynamics (velocity and adhesion). Our revised glycerolization protocol (40% v/v final) resulted in >98.5% RBC recovery following freezing (-80°C) and thawing (37°C), with no difference compared to the standard reported method (40% w/v final). Full deglycerolization (>99.9% glycerol removal) of 40% v/v final samples resulted in total cumulative lysis of ~8%, compared to ~12–15% with the standard method. The post cryopreservation/deglycerolization RBC phenotype was indistinguishable from that for fresh RBCs with regard to physical RBC parameters (morphology, volume, and density), osmotic fragility, deformability, endothelial adhesivity, O2 affinity, vasoregulation, metabolomics, and flow dynamics. These results indicate that RBC cryopreservation/deglycerolization in 40% v/v glycerol final does not significantly impact RBC phenotype (compared to fresh cells).

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<![CDATA[Chain organization of human interphase chromosome determines the spatiotemporal dynamics of chromatin loci]]> https://www.researchpad.co/article/5c0ed753d5eed0c484f13f19

We investigate spatiotemporal dynamics of human interphase chromosomes by employing a heteropolymer model that incorporates the information of human chromosomes inferred from Hi-C data. Despite considerable heterogeneities in the chromosome structures generated from our model, chromatins are organized into crumpled globules with space-filling (SF) statistics characterized by a single universal scaling exponent (ν = 1/3), and this exponent alone can offer a quantitative account of experimentally observed, many different features of chromosome dynamics. The local chromosome structures, whose scale corresponds to that of topologically associated domains (∼ 0.1 − 1 Mb), display dynamics with a fast relaxation time (≲ 1 − 10 sec); in contrast, the long-range spatial reorganization of the entire chromatin (O(102) Mb) occurs on a much slower time scale (≳ hour), providing the dynamic basis of cell-to-cell variability and glass-like behavior of chromosomes. Biological activities, modeled using stronger isotropic white noises added to active loci, accelerate the relaxation dynamics of chromatin domains associated with the low frequency modes and induce phase segregation between the active and inactive loci. Surprisingly, however, they do not significantly change the dynamics at local scales from those obtained under passive conditions. Our study underscores the role of chain organization of chromosome in determining the spatiotemporal dynamics of chromatin loci.

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<![CDATA[Physicochemical and microbiological characterization of the sensory deviation responsible for the origin of the special sherry wines "palo cortado" type]]> https://www.researchpad.co/article/5c1ab857d5eed0c484027a29

The aim of this study was to characterize the biochemical changes and microbiological processes involved in the sensory deviation of “sobretablas” wines during biological aging, which leads to the origin of special or rare “palo cortado” wines. Industrial trials of biological aging of “sobretablas” wines with the potential for the development of lactic acid bacteria (LAB) were performed to study this phenomenon. The results showed that sensory deviation was due to the development of malolactic fermentation (MLF) together with an attenuated aerobic metabolism of “flor” yeast. Malolactic fermentation (MLF) was promoted by the presence of malic acid concentrations that were higher than 1 g/L and the coexistence of LAB and “flor” velum yeast. Ethyl lactate, acetoin and 2,3-butanediol are some of the volatile compounds that are responsible for this sensory deviation. Wines with high levels of gluconic and malic acids (> 1 g/L) can cause, with very low probability, the sensory deviation of “palo cortado”. A lysozyme dose of 12 g/hL is an effective treatment to avoid malolactic fermentation (MFL) and sensory deviation. Understanding the biochemical and microbiological changes involved in sensory deviation can be useful to wineries as markers to identify the origin of the special sherry wines "palo cortado" type.

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<![CDATA[Structural insight into substrate and product binding in an archaeal mevalonate kinase]]> https://www.researchpad.co/article/5c12cf8ed5eed0c484914932

Mevalonate kinase (MK) is a key enzyme of the mevalonate pathway, which produces the biosynthetic precursors for steroids, including cholesterol, and isoprenoids, the largest class of natural products. Currently available crystal structures of MK from different organisms depict the enzyme in its unbound, substrate-bound, and inhibitor-bound forms; however, until now no structure has yet been determined of MK bound to its product, 5-phosphomevalonate. Here, we present crystal structures of mevalonate-bound and 5-phosphomevalonate-bound MK from Methanosarcina mazei (MmMK), a methanogenic archaeon. In contrast to the prior structure of a eukaryotic MK bound with mevalonate, we find a striking lack of direct interactions between this archaeal MK and its substrate. Further, these two MmMK structures join the prior structure of the apoenzyme to complete the first suite of structural snapshots that depict unbound, substrate-bound, and product-bound forms of the same MK. With this collection of structures, we now provide additional insight into the catalytic mechanism of this biologically essential enzyme.

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<![CDATA[Towards a molecular basis of ubiquitin signaling: A dual-scale simulation study of ubiquitin dimers]]> https://www.researchpad.co/article/5bf86f60d5eed0c48405a957

Covalent modification of proteins by ubiquitin or ubiquitin chains is one of the most prevalent post-translational modifications in eukaryotes. Different types of ubiquitin chains are assumed to selectively signal respectively modified proteins for different fates. In support of this hypothesis, structural studies have shown that the eight possible ubiquitin dimers adopt different conformations. However, at least in some cases, these structures cannot sufficiently explain the molecular basis of the selective signaling mechanisms. This indicates that the available structures represent only a few distinct conformations within the entire conformational space adopted by a ubiquitin dimer. Here, molecular simulations on different levels of resolution can complement the structural information. We have combined exhaustive coarse grained and atomistic simulations of all eight possible ubiquitin dimers with a suitable dimensionality reduction technique and a new method to characterize protein-protein interfaces and the conformational landscape of protein conjugates. We found that ubiquitin dimers exhibit characteristic linkage type-dependent properties in solution, such as interface stability and the character of contacts between the subunits, which can be directly correlated with experimentally observed linkage-specific properties.

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<![CDATA[HCV Induces Telomerase Reverse Transcriptase, Increases Its Catalytic Activity, and Promotes Caspase Degradation in Infected Human Hepatocytes]]> https://www.researchpad.co/article/5989da05ab0ee8fa60b75647

Introduction

Telomerase repairs the telomeric ends of chromosomes and is active in nearly all malignant cells. Hepatitis C virus (HCV) is known to be oncogenic and potential interactions with the telomerase system require further study. We determined the effects of HCV infection on human telomerase reverse transcriptase (TERT) expression and enzyme activity in primary human hepatocytes and continuous cell lines.

Results

Primary human hepatocytes and Huh-7.5 hepatoma cells showed early de novo TERT protein expression 2–4 days after infection and these events coincided with increased TERT promoter activation, TERT mRNA, and telomerase activity. Immunoprecipitation studies demonstrated that NS3-4A protease-helicase, in contrast to core or NS5A, specifically bound to the C-terminal region of TERT through interactions between helicase domain 2 and protease sequences. Increased telomerase activity was noted when NS3-4A was transfected into cells, when added to reconstituted mixtures of TERT and telomerase RNA, and when incubated with high molecular weight telomerase ‘holoenzyme’ complexes. The NS3-4A catalytic effect on telomerase was inhibited with primuline or danoprevir, agents that are known to inhibit NS3 helicase and protease activities respectively. In HCV infected cells, NS3-4A could be specifically recovered with telomerase holoenzyme complexes in contrast to NS5A or core protein. HCV infection also activated the effector caspase 7 which is known to target TERT. Activation coincided with the appearance of lower molecular weight carboxy-terminal fragment(s) of TERT, chiefly sized at 45 kD, which could be inhibited with pancaspase or caspase 7 inhibitors.

Conclusions

HCV infection induces TERT expression and stimulates telomerase activity in addition to triggering Caspase activity that leads to increased TERT degradation. These activities suggest multiple points whereby the virus can influence neoplasia. The NS3-4A protease-helicase can directly bind to TERT, increase telomerase activity, and thus potentially influence telomere repair and host cell neoplastic behavior.

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