Mineralization Potential of Polarized Dental Enamel

Background: Management of human teeth has moved from a surgical to a more conservative approach of inhibiting or preventing lesion progression. Increasing enamel mineralization is crucial in this regard. A potential difficulty is the preferential mineralization of the outermost portion of the enamel that can prevent overall mineralization. We describe a strategy for increasing the mineralization potential of dental enamel. Methodology/Principal Findings: Extracted human premolar teeth enamel (n = 5) were exposed to a high concentration of hydrogen peroxide with an energizing source. Samples were stored in artificial saliva at 37uC for 1 wk. A desktop X-ray micro-CT system was used to evaluate the mineral density of samples. Mineral distribution was polarized between the lower and the higher mineralized portion of enamel by charged oxygen free radicals due to activation of permeated hydrogen peroxide. The kinetics of energy absorption in the deeper enamel region demonstrated improvement of preferential mineralization into the region without restricting overall mineralization of the enamel. Subsequent increasing mineralization, even in the dense mineralized outer portion of enamel, was also achieved. Conclusions/Significance: This increased mineralization may promote resistance to acidic deterioration of the structure. The present study is one of the primary steps towards the development of novel application in reparative and restorativ

Non-Invasive Bleaching of the Human Lens by Femtosecond Laser Photolysis

Background: Globally, cataract is the leading cause of blindness and impaired vision. Cataract surgery is an attractive treatment option but it remains unavailable in sufficient quantity for the vast majority of the world population living in areas without access to specialized health care. Reducing blindness from cataract requires solutions that can be applied outside operating theatres. Cataract is a protein conformational disease characterized by accumulation of light absorbing, fluorescent and scattering protein aggregates. The aim of the study was to investigate whether these compounds were susceptible to photobleaching by a non-invasive procedure and whether this would lead to optical rejuvenation of the lens. Methodology/Principal Findings: Nine human donor lenses were treated with an 800 nm infra-red femtosecond pulsed laser in a treatment zone measuring 16160.52 mm. After laser treatment the age-induced yellow discoloration of the lens was markedly reduced and the transmission of light was increased corresponding to an optical rejuvenation of 3 to 7 years. Conclusions/Significance: The results demonstrate that the age-induced yellowing of the human lens can be bleached by a non-invasive procedure based on femtosecond laser photolysis. Cataract is a disease associated with old age. At the current technological stage, lens aging is delayed but with a treatment covering the entire lens volume complete optical rejuvenation is expected. Thus, femtosecond photolysis has the potential clinical value of replacing invasive cataract surgery by a non-invasive treatment modality that can be placed in mobile units, thus breaking down many of the barriers impedin

Amyloid-like protein inclusions in tobacco transgenic plants

The formation of insoluble protein deposits in human tissues is linked to the onset of more than 40 different disorders, ranging from dementia to diabetes. In these diseases, the proteins usually self-assemble into ordered β-sheet enriched aggregates known as amyloid fibrils. Here we study the structure of the inclusions formed by maize transglutaminase (TGZ) in the chloroplasts of tobacco transplastomic plants and demonstrate that they have an amyloid-like nature. Together with the evidence of amyloid structures in bacteria and fungi our data argue that amyloid formation is likely a ubiquitous process occurring across the different kingdoms of life. The discovery of amyloid conformations inside inclusions of genetically modified plants might have implications regarding their use for human applications

Preparative Scale Production of Functional Mouse Aquaporin 4 Using Different Cell-Free Expression Modes

The continuous progress in the structural and functional characterization of aquaporins increasingly attracts attention to study their roles in certain mammalian diseases. Although several structures of aquaporins have already been solved by crystallization, the challenge of producing sufficient amounts of functional proteins still remains. CF (cell free) expression has emerged in recent times as a promising alternative option in order to synthesize large quantities of membrane proteins, and the focus of this report was to evaluate the potential of this technique for the production of eukaryotic aquaporins. We have selected the mouse aquaporin 4 as a representative of mammalian aquaporins. The protein was synthesized in an E. coli extract based cell-free system with two different expression modes, and the efficiencies of two modes were compared. In both, the P-CF (cell-free membrane protein expression as precipitate) mode generating initial aquaporin precipitates as well as in the D-CF (cell-free membrane protein expression in presence of detergent) mode, generating directly detergent solubilized samples, we were able to obtain mg amounts of protein per ml of cell-free reaction. Purified aquaporin samples solubilized in different detergents were reconstituted into liposomes, and analyzed for the water channel activity. The calculated Pf value of proteoliposome samples isolated from the D-CF mode was 133 µm/s at 10°C, which was 5 times higher as that of the control. A reversible inhibitory effect of mercury chloride was observed, which is consistent with previous observations of in vitro reconstituted aquaporin 4. In this study, a fast and convenient protocol was established for functional expression of aquaporins, which could serve as basis for further applications such as water filtration

Designer TGFβ Superfamily Ligands with Diversified Functionality

Transforming Growth Factor – beta (TGFβ) superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs), and Bone Morphogenetic Proteins (BMPs), are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer), to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values

Optimized Expression of Full-Length IgG1 Antibody in a Common E. coli Strain

Multi-polypeptide proteins such as antibodies are difficult to express in prokaryotic systems such as E. coli due to the complexity of protein folding plus secretion. Thus far, proprietary strains or fermenter cultures have been required for appreciable yields. Previous studies have shown that expression of heterologous proteins in E. coli can be enhanced by the reduction of protein translation rates. In this paper, we demonstrate that useful quantities of full-length IgG can be expressed and purified from the common E. coli laboratory strain HB2151 in standard shaking culture using a simple strategy of reduced inducer concentration combined with delayed induction times to modulate translation rates. Purified IgG had only marginally reduced avidity compared to mammalian derived IgG. This indicates that this technique can be used to derive antibodies of potentially equal utility as those expressed in mammalian cell culture, particularly for applications where effector functions mediated by the glycosylated residues in the Fragment Crystallizable (Fc) portion of the immunoglobulin are not required

Bacterial inclusion bodies are industrially exploitable amyloids

Understanding the structure, functionalities and biology of functional amyloids is an issue of emerging interest. Inclusion bodies, namely protein clusters formed in recombinant bacteria during protein production processes, have emerged as unanticipated, highly tunable models for the scrutiny of the physiology and architecture of functional amyloids. Based on an amyloidal skeleton combined with varying amounts of native or native-like protein forms, bacterial inclusion bodies exhibit an unusual arrangement that confers mechanical stability, biological activity and conditional protein release, being thus exploitable as versatile biomaterials. The applicability of inclusion bodies in biotechnology as enriched sources of protein and reusable catalysts, and in biomedicine as biocompatible topographies, nanopills or mimetics of endocrine secretory granules has been largely validated. Beyond these uses, the dissection of how recombinant bacteria manage the aggregation of functional protein species into structures of highly variable complexity offers insights about unsuspected connections between protein quality (conformational status compatible with functionality) and cell physiology.info:eu-repo/semantics/acceptedVersio

Bacterial inclusion bodies are industrially exploitable amyloids

Altres ajuts: CERCA Programme/Generalitat de CatalunyaUnderstanding the structure, functionalities and biology of functional amyloids is an issue of emerging interest. Inclusion bodies, namely protein clusters formed in recombinant bacteria during protein production processes, have emerged as unanticipated, highly tunable models for the scrutiny of the physiology and architecture of functional amyloids. Based on an amyloidal skeleton combined with varying amounts of native or native-like protein forms, bacterial inclusion bodies exhibit an unusual arrangement that confers mechanical stability, biological activity and conditional protein release, being thus exploitable as versatile biomaterials. The applicability of inclusion bodies in biotechnology as enriched sources of protein and reusable catalysts, and in biomedicine as biocompatible topographies, nanopills or mimetics of endocrine secretory granules has been largely validated. Beyond these uses, the dissection of how recombinant bacteria manage the aggregation of functional protein species into structures of highly variable complexity offers insights about unsuspected connections between protein quality (conformational status compatible with functionality) and cell physiology

Non-Conjugated Small Molecule FRET for Differentiating Monomers from Higher Molecular Weight Amyloid Beta Species

Background: Systematic differentiation of amyloid (Aβ) species could be important for diagnosis of Alzheimer's disease (AD). In spite of significant progress, controversies remain regarding which species are the primary contributors to the AD pathology, and which species could be used as the best biomarkers for its diagnosis. These controversies are partially caused by the lack of reliable methods to differentiate the complicated subtypes of Aβ species. Particularly, differentiation of Aβ monomers from toxic higher molecular weight species (HrMW) would be beneficial for drug screening, diagnosis, and molecular mechanism studies. However, fast and cheap methods for these specific aims are still lacking. Principal Findings: We demonstrated the feasibility of a non-conjugated FRET (Förster resonance energy transfer) technique that utilized amyloid beta (Aβ) species as intrinsic platforms for the FRET pair assembly. Mixing two structurally similar curcumin derivatives that served as the small molecule FRET pair with Aβ40 aggregates resulted in a FRET signal, while no signal was detected when using Aβ40 monomer solution. Lastly, this FRET technique enabled us to quantify the concentrations of Aβ monomers and high molecular weight species in solution. Significance: We believe that this FRET technique could potentially be used as a tool for screening for inhibitors of Aβ aggregation. We also suggest that this concept could be generalized to other misfolded proteins/peptides implicated in various pathologies including amyloid in diabetes, prion in bovine spongiform encephalopathy, tau protein in AD, and α-synuclein in Parkinson disease.National Institute on Aging (K25AG036760

Identification and Localization of Proteins Associated with Biomineralization in the Iron Deposition Vesicles of Honeybees (Apis mellifera)

Honeybees (Apis mellifera) form superparamagnetic magnetite to act as a magnetoreceptor for magnetoreception. Biomineralization of superparamagnetic magnetite occurs in the iron deposition vesicles of trophocytes. Even though magnetite has been demonstrated, the mechanism of magnetite biomineralization is unknown. In this study, proteins in the iron granules and iron deposition vesicles of trophocytes were purified and identified by mass spectrometry. Antibodies against such proteins were produced. The major proteins include actin, myosin, ferritin 2, and ATP synthase. Immunolabeling and co-immunoprecipitation studies suggest that iron is stored in ferritin 2 for the purpose of forming 7.5-nm diameter iron particles and that actin-myosin-ferritin 2 may serve as a transporter system. This system, along with calcium and ATP, conveys the iron particles (ferritin) to the center of iron deposition vesicles for iron granules formation. These proteins and reactants are included in iron deposition vesicles during the formation of iron deposition vesicles from the fusion of smooth endoplasmic reticulum. A hypothetical model for magnetite biomineralization in iron deposition vesicles is proposed for honeybees