1,720,997 research outputs found
Selective Recovery of Rare Earth Elements from Wastes Using Engineered Proteins
No full textRare earth elements (REEs) have become increasingly important materials owing to their use in the high-tech and clean-energy industries. However, the unpredictable supply, possible health risks, and environmentally unsustainable extraction practices associated with REEs have encouraged the development of green technologies for the selective extraction and recovery of metals. This study presents a simple and innovative approach for the selective extraction and recovery of total REEs. Elastin-like polypeptide (ELP) and the REE-binding domain (lanmodulin) are fused to form REEs-sensitive and thermo-responsive genetically encoded ELP called RELP, where ELP offered a reversible, inverse phase transition for repeated uses. The RELP are purified and used for the selective extraction of total REEs from competing non-REEs metals by controlling the solution temperature (4 and 37 °C) and pH. RELP exhibit high REE specificity, even in the presence of non-REE metal ions. The bound REEs are readily recovered during at least six repeated cycles, and the efficiency is maintained. Moreover, REEs are selectively recovered by RELP from steel slag leachate, a potential industrial source of REEs. RELP offers a rapid, selective, and scalable method for REE extraction and recovery. This technology can be adapted to recover other precious metals and commodities. Rare-earth elements (REEs), including the lanthanide series, are crucial components essential for clean energy transitions and originate from geographically limited regions.
Exploiting new and more diverse supply sources is imperative to facilitate a clean energy future. Hence, we have explored the recovery of REEs from coal fly ash, a complex, low-grade industrial feedstock that is currently underutilized. Specifically, we demonstrated the thermo-responsive genetically encoded elastin-like polypeptide fused with lanmodulin (RELP) as a recyclable bioengineered protein adsorbent for the selective retrieval of REEs from coal fly ash, which typically contains very low REE levels. RELP efficiently achieves the selective recovery of REEs from fly ash by inducing a phase transition from a solution to a coacervate state through a series of heat-cool cycles (below and above the transition temperature). Successful selective recovery has been demonstrated over multiple cycles. Additionally, RELP has the capability to produce high-purity REEs from sources with extremely low REE concentrations. This study offers a sustainable approach to diversify REE supplies by environmentally friendly management of REE-containing waste and addresses a critical research need for its applications in bio-extracting REEs from fly ash.
Lanmodulin is becoming an emerging biological chelator for REEs binding protein. Previous research showed that covalent immobilization of Lanmodulin on supporting materials can be explored as an effective strategy to construct robust and reusable biomaterials for the selective recovery of REEs. However, there are drawbacks associated with these methods as these studies required complex material synthesis and functionalization processes, resulting in reduced protein loading capacity and REEs adsorption activity. There is growing interest in using solid-binding peptides and proteins (SBPs), which recognize and bind to solid surfaces via non-covalent interactions. Here, LanM-PS, a novel recombinant protein, was prepared. PS is a polystyrene binding tag that can specifically bind polystyrene. The biosorbent polystyrene-LanM-PS was obtained by binding of LanM-PS to polystyrene materials. The biosorbent showed adsorption for REEs. With further studies, this environmentally friendly biosorbent can be used as a cost-effective, non-toxic, and reusable material for REEs recovery in batch and continuous operation from low-grade feedstocks.Docto
Synthesis and characterization of the metal coated PVP microneedle patch for drug delivery system using thermal or electrical stimulation
No full textA conventional microneedle patch has the disadvantages of weak mechanical properties and low drug-release efficiency when using polymers such as hyaluronic acid, chitosan, cellulose, and polycaprolactone. In this study, we prepared a novel microneedle patch using metal bioelectrodes that provide excellent mechanical properties, drug-delivery efficiency, and biocompatibility at the same time. This biocompatible polyvinylpyrrolidone (PVP) based hydrogel-type microneedle patch was prepared using gamma rays to compensate for its weak mechanical properties. The back side of the PVP based microneedle patch were coated with gold or silver (which provides excellent thermal and electrical conductivity) as bioelectrodes using the thermal evaporation method to maximize or control the efficiency of drug release. The metal (Au or Ag)-coated microneedle patch has enough mechanical properties to successfully penetrate the skin. Even after being fixed to the skin, the shape of a needle tip was hardly deformed, and exhibited resistance to breakage even when bent due to its flexibility. In addition, it was confirmed using a Franz cell that the amount of drug released from a drug-loaded microneedle patch was maximized by thermal or electrical stimulus. The drug-release efficiency increased more than 7.9 times (790%) compared to a flat PVP patch (without applying electricity) when 3 V was applied. In addition, when ~ 40 ℃ heat were applied, the drug-release efficiency of the Au- and Ag-coated PVP based microneedle patches improved about 5.3 times (530%) compared to a flat PVP patch at room temperature. These excellent properties of our metal-coated PVP based microneedle patches can be used not only in whitening or anti-wrinkling cosmetics, but also as medical drug-delivery systems to treat such as hypertension, diabetes, and arthritis.Maste
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Rare earth elements recovery using elastin like polypeptide fused with lanmodulin from fly ash
No full textDue to increasing significance of rare earth elements (REEs) in modern era for environment friendly and high tech technologies, there’s a tremendous demand for REEs recovery from the wastes in a sustainable way to avoid the environmental burden. In this regard, REEextractor technology was developed by Zohaib Hussain for capturing REEs using elastin like polypeptide fused with Lanmodulin (RELP) in pH responsive manner. In this study, fly ash which is a by product of coal incineration in a coal thermal plant, is utilized for the REEs recovery about 98% total REEs recovery at ph 4.02 and 5.7 with minimal Cu recovery. Moreover, the enrichment of REEs was also briefly investigated. Therefore, REEextractor has a great potential to recover and enrich REEs from various types of industrial and electronic wastes.Maste
A Novel Albumin Conjugation Platform via Covalent Protein Ligation for Long-Acting Therapeutic Proteins
No full textTherapeutic proteins, pivotal for treating diverse human diseases, often face challenges such as rapid serum clearance due to renal filtration and enzymatic degradation. To address this issue and enable prolonged therapeutic efficacy, techniques to extend the serum half-life of therapeutic proteins are crucial. Human serum albumin (HSA) has been used as a half-life extender due to its long intrinsic half-life attributed to Fc receptor (FcRn)-mediated recycling. In this study, we employed the SpyTag/SpyCatcher (ST/SC) system, known for forming irreversible isopeptide bonds, to conjugate HSA and therapeutic proteins. The AlbuCatcher platform, a conjugate of HSA and SC, was proposed aiming to provide an easy method to conjugate an ST-fused therapeutic protein to HSA. To minimize activity loss, site-specific HSA conjugation to SC was achieved using the inverse electron-demand Diels-Alder (IEDDA) reaction. Using urate oxidase (Uox) as a model protein with a short half-life, ST was fused to generate Uox-ST. Then, HSA-conjugated Uox (Uox-HSA) was successfully prepared via the Uox-ST/AlbuCatcher reaction, demonstrating a substantial reaction yield (>95%). The in vitro enzyme activity of Uox-ST and Uox-HSA conjugate was comparable to that of unmodified Uox. Moreover, pharmacokinetics studies in mice revealed the serum half-life of the Uox-HSA conjugate to be ~18 h, which was 9.8 h longer than that of the native Uox. This extended half-life clearly shows the effectiveness of the AlbuCatcher platform in enhancing therapeutic protein pharmacokinetics.Maste
Extending Serum Half-life in Vivo of Therapeutic Protein using Albumin-Binding Ligands
No full textTherapeutic proteins are indispensable for the treatment of various human diseases. However, intrinsic short serum half-lives of proteins remain significant hurdles for developing new therapeutic proteins or expanding applications of existing ones. In the case of urate oxidase (Uox), a therapeutic protein used for the treatment of hyperuricemia, due to its short half-life, its application for gout treatment requires prolongation of its half-life in vivo.
First, conjugation of a fatty acid (FA), a human serum albumin (HSA) ligand, to therapeutic proteins/peptides is an emerging strategy to prolong serum protein half-life via neonatal Fc receptor (FcRn)–mediated recycling. Fatty acid conjugation is effective for peptides and small proteins (less than 28 kDa), but not for Uox (140 kDa). I hypothesized that 1) the intramolecular distance in the conjugate of FA and Uox is a critical factor for effective FcRn-mediated recycling, and 2) small proteins do not cause substantial competition for FcRn binding to albumin, resulting in an extended serum half-life. To control the intramolecular distance in the conjugate, I varied linker lengths between Uox and palmitic acid (PA). These results demonstrate that the intramolecular distance in the conjugate of Uox and FA governs the stable formation of FcRn/HSA/FA-conjugated protein and serum half-life extension in vivo. Unlike the FA-conjugated large protein, the small proteins conjugated with the same set of linkers exhibited comparable FcRn binding to albumin and the extended serum half-lives.
Second, injectable hydrogels were used as carriers for the controlled release of therapeutic proteins as well as for convenient spatial release control. However, native interactions between therapeutic proteins and hydrogels are often not strong enough for extended-release of therapeutic proteins. Therefore, implementing selective and strong interactions with hydrogels and therapeutic proteins is required. Herein, I investigated whether strong and selective interactions between HSA and albumin binding peptide (ABP) could be used to achieve a substantially extended release of therapeutic protein from injectable gels. The serum activity half-life of Uox-HSA conjugate injected with the ABP-outfitted PEG-PAEU hydrogels was 96 h, which was 88 times longer than that of the native Uox without any type of hydrogel carrier.
Third, in order to improve conjugation yield and biocompatibility of the injectable gel delivery system, I chose the elastin-like polypeptide (ELP) as a based material for delivery vehicle preparation. ELP is a biopolymer consisting of a repeated [V‐P‐G‐X‐G, where X can be any amino acid except proline] pentapeptide sequences inspired by native human elastin. ELP has been investigated as in situ‐forming and injectable depots for drug delivery. Herein, the objective of this study was to develop a biocompatible, injectable, and genetically encodable ELP coacervate that could provide for sustained release of a therapeutic protein. As a result, HSA-conjugated therapeutic protein carried by the albumin ligand-conjugated ELP coacervate exhibits improved sustained-release and extended half-life.
The significance of this dissertation research lies in successful prolongation of the serum half-life of Uox using an appropriate linker between FA and Uox. In addition, our results supported that the protein size in the FA-protein conjugate is a crucial factor for effective binding of FcRn with serum albumin, leading to protracted serum half-life in vivo. I believe my findings can contribute to the successful design of other FA conjugated therapeutic proteins for serum half-life extension. Based on ABP-conjugated injectable hydrogels with strong HSA affinity, this approach may also be applied to other albumin-based therapeutic models for clinical applications. Finally, it was demonstrated that albumin binding ligand-conjugated ELP coacervate can served biocompatible, injectable, and genetically encodable polypeptide based carrier for drug delivery.Docto
Controlling the Release Rate of Therapeutic Proteins from Injectable Alginate Hydrogels Using Charge-Charge Interactions
No full textGenerally, therapeutic proteins have a short half-life. To complement this, methods are used to induce sustained release of the protein. A typical approach is to deliver the protein by encapsulating it in a functionalized delivery carrier. However, this method is complex as it requires multiple reactions. Therefore, this study demonstrates that the release rate can be controlled by inducing charge-charge interaction between the protein and the delivery carrier. Therapeutic proteins that introduce highly charged peptides and delivery carriers with opposite charges can interact to cause slow release. This method slows the release rate by 2.3 times in vitro. Thus, introducing charged short peptides into therapeutic proteins can be used as a simple and systematic method to control the release rate.Maste
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Site-specific bioconjugation of proteins for in vivo applications
No full textBioconjugation of proteins has been used as the breakthrough approach to biological applications. In particular, recent advanced site-specific bioconjugation has led to the development of methods for various applications, including drug delivery and diagnosis. In order to achieve these approaches, biorthogonal chemistry and site-specific unnatural amino acids (UAAs) containing biorthogonal reactive handles were developed. So far, over 200 UAAs containing click chemistry reactive handles were successfully incorporated into protein and applied to extend the function of proteins without perturbation of their function and structure. Therefore, site-specific bioconjugation of protein has a great potential for biological applications. Here, I investigated site-specific bioconjugations of proteins using optimization of reactivity and stability for efficient in vivo applications.
In Chapter Ⅱ, I investigated genetically encoded a phenylalanine analog containing a hydrogen-substituted tetrazine (frTet) and determined its ability to increase the IEDDA reaction rate and allow successful bioconjugation in vivo. The IEDDA reaction rate of superfolder green fluorescent protein (sfGFP) containing frTet (sfGFP-frTet) was 12-fold greater than that of sfGFP containing methyl-substituted tetrazine (sfGFP-Tet_v2.0) in vitro. Moreover, sfGFP-frTet encapsulated with pluronic-based nanocarriers, and transferred into nude mice or tumor-bearing mice for in vivo imaging revealed fluorescence recovery upon addition of trans- cyclooctene via the IEDDA reaction. These results demonstrated that the genetically encoded frTet allows an almost complete IEDDA reaction in vivo upon addition of trans-cyclooctene, enabling temporal control of in vivo bioconjugation in high yield.
In Chapter Ⅲ, I investigated species-dependent albumin-FcRn interactions using site-specific conjugation of species-matched albumin (mouse serum albumin, MSA) and species-mismatched albumin (human serum albumin, HSA) into sfGFP (superfolder green fluorescence protein). The clickable unnatural amino acid containing p-azido phenylalanine was site-specifically introduced into a sfGFP followed by conjugation to an albumin species via a hetero-bifunctional linker and SPAAC (strain promoted azide-alkyne cycloaddition). Then, sfGFP-MSA and HSA were directly compared of serum half-life in non-transgenic mice. Conjugation of HSA led to very limited extension of the serum half-life of sfGFP in mice (16.3 h), compared to that of HSA in transgenic mice harboring an allele of mouse FcRn knock-out and expressing human FcRn (67 h). However, conjugation of mouse serum albumin (MSA) resulted in a serum half-life of sfGFP (27.7 h) comparable to that of MSA in mice (28.8 h). Therefore, these results supported that albumin–FcRn interactions are species dependent in vivo.
In Chapter Ⅳ, I investigated that conjugation of multiple HSA molecules to a therapeutic protein significantly further extends the serum half-life via multivalent HSA-FcRn interactions. I chose urate oxidase (Uox), a tetrameric therapeutic protein used for the treatment of gout, as a model protein. And frTet was site-specifically introduced into position 174 of each subunit of Uox. The four HSA molecules (Uox-HSA4), one or two HSA molecules (Uox-HSA1 or Uox-HSA2) were prepared via IEDDA and SPAAC, respectively. The enzyme activity of all three Uox-HSA conjugates was comparable to that of unmodified Uox. And I found out that an increase in HSA molecules conjugated to Uox (multiple albumin-conjugated therapeutic protein) enhanced FcRn binding and consequently prolonged the serum half-life in vivo. In particular, the conjugation of four HSA molecules to Uox led to a prominent extension of serum half-life (over 21 h), which is about 16-fold longer than that of Uox-WT.
In Chapter Ⅴ, in order to develop a thermostable and long-circulating urate oxidase, I chose urate oxidase originating from Arthrobacter globiformis (AgUox), which has been reported to be thermostable and less immunogenic than the Aspergillus form. Then I investigated the site-specific conjugation of HSA to AgUox based on the site-specific incorporation of a clickable unnatural amino acid (frTet) and inverse electron demand Diels-Alder reaction (IEDDA). AgUox containing frTet at position 196 (AgUox_196frTet) exhibited enzymatic activity and thermostability comparable to those of wild-type AgUox. Furthermore, AgUox_196frTet exhibited a high HSA conjugation yield without compromising its enzymatic activity, generating well-defined HSA-conjugated AgUox (AgUox_196HSA). In mice, the serum half-life of AgUox_196HSA was approximately 29 h, or roughly 16 times longer than that of wild-type AgUox.
In Chapter Ⅵ, in order to improve the in vivo stability of albumin-conjugated therapeutic protein, I introduced the APN chemistry into the site-specific albumin conjugation method. To demonstrate the effectiveness of APN chemistry, I performed site-specific human serum albumin (HSA) conjugation into Urate oxidase originating from Arthrobacter globiformis using thiol-maleimide or -APN chemistry and then directly compared the serum half-life of conjugates. The substantial cleavage of thioester of AgUox-MAL-HSA was observed in vitro, whereas no cleavage of thiol-APN product of AgUox-APN-HSA was observed. Furthermore, the in vivo serum half-life of AgUox-APN-HSA in the late phase was significantly longer than that of AgUox-MAL-HSA indicating the enhanced in vivo stability of AgUox-APN-HSA. Overall, these results demonstrate that the thiol-APN chemistry enhanced the in vivo stability of HSA-conjugated therapeutic protein.
This dissertation research lies in the investigation of site-specific bioconjugation of proteins for its applications in vivo. To achieve this purpose, I performed with site-specific incorporation of UAAs containing biorthogonal reactive handles into proteins and generating well-defined protein conjugates by bioconjugation chemistries. And the effectiveness of site-specific bioconjugation of proteins in vivo was evaluated by in vivo imaging and pharmacokinetic studies.Docto
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