In a randomized trial, 6545 patients with acute heart failure were assigned to either serelaxin or placebo in addition to standard care. There were no significant differences between the two groups ...in the incidence of death from cardiovascular causes at 180 days or worsening heart failure at 5 days.
Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in ...multiple sclerosis in humans. Etx is a member of the aerolysin family of β-PFTs (aβ-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double β-barrel, a common feature of the aβ-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.
α-Synuclein (aSyn) fibrillar polymorphs have distinct in vitro and in vivo seeding activities, contributing differently to synucleinopathies. Despite numerous prior attempts, how polymorphic aSyn ...fibrils differ in atomic structure remains elusive. Here, we present fibril polymorphs from the full-length recombinant human aSyn and their seeding capacity and cytotoxicity in vitro. By cryo-electron microscopy helical reconstruction, we determine the structures of the two predominant species, a rod and a twister, both at 3.7 Å resolution. Our atomic models reveal that both polymorphs share a kernel structure of a bent β-arch, but differ in their inter-protofilament interfaces. Thus, different packing of the same kernel structure gives rise to distinct fibril polymorphs. Analyses of disease-related familial mutations suggest their potential contribution to the pathogenesis of synucleinopathies by altering population distribution of the fibril polymorphs. Drug design targeting amyloid fibrils in neurodegenerative diseases should consider the formation and distribution of concurrent fibril polymorphs.
Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signaling cascades, enabling them to stably ...maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signaling molecules (PP-InsPs), which are sensed by SPX domain-containing proteins. In plants, PP-InsP-bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP
-SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP
regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors.
More than 70 years ago, the filamentous ascomycete Trichoderma reesei was isolated on the Solomon Islands due to its ability to degrade and thrive on cellulose containing fabrics. This trait that ...relies on its secreted cellulases is nowadays exploited by several industries. Most prominently in biorefineries which use T. reesei enzymes to saccharify lignocellulose from renewable plant biomass in order to produce biobased fuels and chemicals. In this review we summarize important milestones of the development of T. reesei as the leading production host for biorefinery enzymes, and discuss emerging trends in strain engineering. Trichoderma reesei has very recently also been proposed as a consolidated bioprocessing organism capable of direct conversion of biopolymeric substrates to desired products. We therefore cover this topic by reviewing novel approaches in metabolic engineering of T. reesei.
Designed ankyrin repeat proteins (DARPins) can recognize targets with specificities and affinities that equal or surpass those of antibodies, but because of their robustness and extreme stability, ...they allow a multitude of more advanced formats and applications. This review highlights recent advances in DARPin design, illustrates their properties, and gives some examples of their use. In research, they have been established as intracellular, real-time sensors of protein conformations and as crystallization chaperones. For future therapies, DARPins have been developed by advanced, structure-based protein engineering to selectively induce apoptosis in tumors by uncoupling surface receptors from their signaling cascades. They have also been used successfully for retargeting viruses. In ongoing clinical trials, DARPins have shown good safety and efficacy in macular degeneration diseases. These developments all ultimately exploit the high stability, solubility, and aggregation resistance of these molecules, permitting a wide range of conjugates and fusions to be produced and purified.
The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked ...glucoses by β-1,4 bonds. The enzyme β-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A β-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a β-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa β-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated β-1,4-endoglucanase had higher activity and stability.
•A strain of Bacillus subtilis with endoglucanase activity was isolated from the wastewater of a pulp and paper mill.•The endoglucanase gene was inserted into a modified plasmid (E. coli) and into pYD1 (S. cerevisiae).•The endoglucanase in pYD1 is expressed with good activity in E. coli, being the first report of this finding.•A truncated recombinant endoglucanase is obtained and is very stable at pH (2-10) and temperature (30–55 °C).
Epstein-Barr virus (EBV) represents a major global health problem. Though it is associated with infectious mononucleosis and ∼200,000 cancers annually worldwide, a vaccine is not available. The major ...target of immunity is EBV glycoprotein 350/220 (gp350) that mediates attachment to B cells through complement receptor 2 (CR2/CD21). Here, we created self-assembling nanoparticles that displayed different domains of gp350 in a symmetric array. By focusing presentation of the CR2-binding domain on nanoparticles, potent neutralizing antibodies were elicited in mice and non-human primates. The structurally designed nanoparticle vaccine increased neutralization 10- to 100-fold compared to soluble gp350 by targeting a functionally conserved site of vulnerability, improving vaccine-induced protection in a mouse model. This rational approach to EBV vaccine design elicited potent neutralizing antibody responses by arrayed presentation of a conserved viral entry domain, a strategy that can be applied to other viruses.
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•Self-assembling nanoparticles present the conserved gp350 receptor-binding domain•The nanoparticles elicit more potent neutralizing antibodies than soluble gp350•These neutralizing antibodies predominantly target the CR2-binding site on gp350•The nanoparticles elicit potent neutralizing antibodies in mice and non-human primates
Structurally designed EBV vaccine candidates based on self-assembling nanoparticles elicit potent and durable virus-neutralizing antibodies that target the receptor-binding site on the viral envelope protein gp350, a site of vulnerability, serving as a template to develop an EBV vaccine and providing a basis for immunofocusing through rational vaccine design.
Human serum albumin: From bench to bedside Fanali, Gabriella; di Masi, Alessandra; Trezza, Viviana ...
Molecular aspects of medicine,
06/2012, Volume:
33, Issue:
3
Journal Article
Peer reviewed
Human serum albumin (HSA), the most abundant protein in plasma, is a monomeric multi-domain macromolecule, representing the main determinant of plasma oncotic pressure and the main modulator of fluid ...distribution between body compartments. HSA displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds. Indeed, HSA represents the main carrier for fatty acids, affects pharmacokinetics of many drugs, provides the metabolic modification of some ligands, renders potential toxins harmless, accounts for most of the anti-oxidant capacity of human plasma, and displays (pseudo-)enzymatic properties. HSA is a valuable biomarker of many diseases, including cancer, rheumatoid arthritis, ischemia, post-menopausal obesity, severe acute graft-versus-host disease, and diseases that need monitoring of the glycemic control. Moreover, HSA is widely used clinically to treat several diseases, including hypovolemia, shock, burns, surgical blood loss, trauma, hemorrhage, cardiopulmonary bypass, acute respiratory distress syndrome, hemodialysis, acute liver failure, chronic liver disease, nutrition support, resuscitation, and hypoalbuminemia. Recently, biotechnological applications of HSA, including implantable biomaterials, surgical adhesives and sealants, biochromatography, ligand trapping, and fusion proteins, have been reported. Here, genetic, biochemical, biomedical, and biotechnological aspects of HSA are reviewed.
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