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1.
Environ Sci Technol ; 57(48): 19902-19911, 2023 Dec 05.
Article in English | MEDLINE | ID: mdl-37983372

ABSTRACT

As global demands for rare-earth elements (REEs) continue to grow, the biological recovery of REEs has been explored as a promising strategy, driven by potential economic and environmental benefits. It is known that calcium-binding domains, including helix-loop-helix EF hands and repeats-in-toxin (RTX) domains, can bind lanthanide ions due to their similar ionic radii and coordination preference to calcium. Recently, the lanmodulin protein from Methylorubrum extorquens was reported, which has evolved a high affinity for lanthanide ions over calcium. Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile, which has been explored for use in bioleaching for metal recovery. In this report, A. ferrooxidans was engineered for the recombinant intracellular expression of lanmodulin. In addition, an RTX domain from the adenylate cyclase protein of Bordetella pertussis, which has previously been shown to bind Tb3+, was expressed periplasmically via fusion with the endogenous rusticyanin protein. The binding of lanthanides (Tb3+, Pr3+, Nd3+, and La3+) was improved by up to 4-fold for cells expressing lanmodulin and 13-fold for cells expressing the RTX domains in both pure and mixed metal solutions. Interestingly, the presence of lanthanides in the growth media enhanced protein expression, likely by influencing protein stability. Both engineered cell lines exhibited higher recoveries and selectivities for four tested lanthanides (Tb3+, Pr3+, Nd3+, and La3+) over non-REEs (Fe2+ and Co2+) in a synthetic magnet leachate, demonstrating the potential of these new strains for future REE reclamation and recycling applications.


Subject(s)
Acidithiobacillus , Lanthanoid Series Elements , Metals, Rare Earth , Calcium/metabolism , Acidithiobacillus/genetics , Acidithiobacillus/chemistry , Acidithiobacillus/metabolism , Lanthanoid Series Elements/metabolism , Ions/metabolism
2.
Biotechnol Rep (Amst) ; 38: e00789, 2023 Jun.
Article in English | MEDLINE | ID: mdl-36923508

ABSTRACT

Cell adhesion is generally a prerequisite to the microbial bioleaching of sulfide minerals, and surface biofilm formation is modulated via quorum sensing (QS) communication. We explored the impact of the overexpression of endogenous QS machinery on the covellite bioleaching capabilities of Acidithiobacillus ferrooxidans, a representative acidophilic chemolithoautotrophic bacterium. Cells were engineered to overexpress the endogenous qs-I operon or just the afeI gene under control of the tac promoter. Both strains exhibited increased transcriptional gene expression of afeI and improved cell adhesion to covellite, including increased production of extracellular polymeric substances and increased biofilm formation. Under low iron conditions, the improved bioleaching of covellite was more evident when afeI was overexpressed alone as compared to the native operon. These observations demonstrate the potential for the genetic modulation of QS as a mechanism for increasing the bioleaching efficiency of covellite, and potentially other copper sulfide minerals.

3.
BMJ Open ; 13(3): e068204, 2023 03 17.
Article in English | MEDLINE | ID: mdl-36931669

ABSTRACT

The COVID-19 pandemic has significantly impacted on the delivery of clinical trials in the UK, posing complicated organisational challenges and requiring adaptations, especially to exercise intervention studies based in the community. We aim to identify the challenges of public involvement, recruitment, consent, follow-up, intervention and the healthcare professional delivery aspects of a feasibility study of exercise in hypertensive primary care patients during the COVID-19 pandemic. While these challenges elicited many reactive changes which were specific to, and only relevant in the context of 'lockdown' requirements, some of the protocol developments that came about during this unprecedented period have great potential to inform more permanent practices for carrying out this type of research. To this end, we detail the necessary adaptations to many elements of the feasibility study and critically reflect on our approach to redesigning and amending this ongoing project in order to maintain its viability to date. Some of the more major protocol adaptations, such as moving the study to remote means wherever possible, had further unforeseen and undesirable outcomes (eg, additional appointments) with regards to extra resources required to deliver the study. However, other changes improved the efficiency of the study, such as the remote informed consent and the direct advertising with prescreening survey. The adaptations to the study have clear links to the UK Plan for the future of research delivery. It is intended that this specific documentation and critical evaluation will help those planning or delivering similar studies to do so in a more resource efficient and effective way. In conclusion, it is essential to reflect and respond with protocol changes in the current climate in order to deliver clinical research successfully, as in the case of this particular study.


Subject(s)
COVID-19 , Hypertension , Humans , COVID-19/epidemiology , SARS-CoV-2 , Pandemics/prevention & control , Feasibility Studies , Exercise , Hypertension/therapy
4.
ChemistryOpen ; 12(1): e202200196, 2023 01.
Article in English | MEDLINE | ID: mdl-36599689

ABSTRACT

A hydrometallurgical process is developed to lower the costs of copper production and thereby sustain the use of copper throughout the global transition to renewable energy technologies. The unique feature of the hydrometallurgical process is the reductive treatment of chalcopyrite, which is in contrast to the oxidative treatment more commonly pursued in the literature. Chalcopyrite reduction by chromium(II) ion is described for the first time and superior kinetics are shown. At high concentrate loadings of 39, 78, and 117 g L-1 , chalcopyrite reacted completely within minutes at room temperature and pressure. The XRD, SEM-EDS, and XPS measurements indicate that chalcopyrite reacts to form copper(I) chloride (CuCl). After the reductive treatment, the mineral products are leached by iron(III) sulfate to demonstrate the complete extraction of copper. The chromium(II) ion may be regenerated by an electrolysis unit inspired by an iron chromium flow battery in a practical industrial process.


Subject(s)
Chlorides , Copper , Ferric Compounds , Chromium , Iron
5.
Nano Lett ; 22(6): 2521-2528, 2022 Mar 23.
Article in English | MEDLINE | ID: mdl-35254075

ABSTRACT

Because it has been demonstrated to be effective toward faster ion diffusion inside the pore space, low-tortuosity porous architecture has become the focus in thick electrode designs, and other possibilities are rarely investigated. To advance current understanding in the structure-affected electrochemistry and to broaden horizons for thick electrode designs, we present a gradient electrode design, where porous channels are vertically aligned with smaller openings on one end and larger openings on the other. With its 3D morphology carefully visualized by Raman mapping, the electrochemical properties between opposite orientations of the gradient electrodes are compared, and faster energy storage kinetics is found in larger openings and more concentrated active material near the separator. As further verified by simulation, this study on gradient electrode design deepens the knowledge of structure-related electrochemistry and brings perspectives in high-energy battery electrode designs.

6.
ACS Appl Mater Interfaces ; 14(8): 10125-10133, 2022 Mar 02.
Article in English | MEDLINE | ID: mdl-35170950

ABSTRACT

Metal processing using microorganisms has many advantages including the potential for reduced environmental impacts as compared to conventional technologies.Acidithiobacillus ferrooxidansis an iron- and sulfur-oxidizing chemolithoautotroph that is known to participate in metal bioleaching, and its metabolic capabilities have been exploited for industrial-scale copper and gold biomining. In addition to bioleaching, microorganisms could also be engineered for selective metal binding, enabling new opportunities for metal bioseparation and recovery. Here, we explored the ability of polyhistidine (polyHis) tags appended to two recombinantly expressed endogenous proteins to enhance the metal binding capacity of A. ferrooxidans. The genetically engineered cells achieved enhanced cobalt and copper binding capacities, and the Langmuir isotherm captures their interaction behavior with these divalent metals. Additionally, the cellular localization of the recombinant proteins correlated with kinetic modeling of the binding interactions, where the outer membrane-associated polyHis-tagged licanantase peptide bound the metals faster than the periplasmically expressed polyHis-tagged rusticyanin protein. The selectivity of the polyHis sequences for cobalt over copper from mixed metal solutions suggests potential utility in practical applications, and further engineering could be used to create metal-selective bioleaching microorganisms.


Subject(s)
Acidithiobacillus , Membrane Proteins , Acidithiobacillus/chemistry , Acidithiobacillus/genetics , Acidithiobacillus/metabolism , Cations, Divalent , Copper/metabolism , Histidine , Membrane Proteins/metabolism
7.
Pilot Feasibility Stud ; 7(1): 192, 2021 Oct 28.
Article in English | MEDLINE | ID: mdl-34711266

ABSTRACT

BACKGROUND: Hypertension  (HTN) affects approximately 25% of the UK population and is a leading cause of mortality. Associated annual health care costs run into billions. National treatment guidance includes initial lifestyle advice, followed by anti-hypertensive medication if blood pressure (BP) remains high. However, adoption and adherence to recommended exercise guidelines, dietary advice and anti-hypertensive medication is poor. Four short bouts of isometric exercise (IE) performed 3 days per week (d/wk) at home elicits clinically significant reductions in BP in those with normal to high-normal BP. This study will determine the feasibility of delivering personalised IE to patients with stage 1 hypertension for whom lifestyle changes would be recommended before medication within NHS primary care. METHODS: This is a randomised controlled feasibility study. Participants were 18+ years, with stage 1 hypertension, not on anti-hypertensive medication and without significant medical contraindications. Trial arms will be standard lifestyle advice (control) or isometric wall squat exercise and standard lifestyle advice. Primary outcomes include the feasibility of healthcare professionals to deliver isometric exercise prescriptions in a primary care NHS setting and estimation of the variance of change in systolic BP. Secondary outcomes include accuracy of protocol delivery, execution of and adherence to protocol, recruitment rate, attrition, perception of intervention viability, cost, participant experience and accuracy of home BP. The study will last 18 months. Sample size of 100 participants (50 per arm) allows for 20% attrition and 6.5% incomplete data, based upon 74 (37 each arm) participants (two-sided 95% confidence interval, width of 1.33 and standard deviation of 4) completing 4 weeks. Ethical approval IRAS ID is 274676. DISCUSSION: Before the efficacy of this novel intervention to treat stage 1 hypertension can be investigated in any large randomised controlled trial, it is necessary to ascertain if it can be delivered and carried out in a NHS primary care setting. Findings could support IE viability as a prophylactic/alternative treatment option. TRIAL REGISTRATION: ISRCTN13472393 , registered 18 August 2020.

8.
Appl Environ Microbiol ; 87(20): e0151821, 2021 09 28.
Article in English | MEDLINE | ID: mdl-34347521

ABSTRACT

Acidithiobacillus ferrooxidans is a well-studied iron- and sulfur-oxidizing acidophilic chemolithoautotroph that is exploited for its ability to participate in the bioleaching of metal sulfides. Here, we overexpressed the endogenous glutamate-cysteine ligase and glutathione synthetase genes in separate strains and found that glutathione synthetase overexpression increased intracellular glutathione levels. We explored the impact of pH on the halotolerance of iron oxidation in wild-type and engineered cultures. The increase in glutathione allowed the modified cells to grow under salt concentrations and pH conditions that are fully inhibitory to wild-type cells. Furthermore, we found that improved iron oxidation ability in the presence of chloride also resulted in higher levels of intracellular reactive oxygen species (ROS) in the strain. These results indicate that glutathione overexpression can be used to increase halotolerance in A. ferrooxidans and would likely be a useful strategy on other acidophilic bacteria. IMPORTANCE The use of acidophilic bacteria in the hydrometallurgical processing of sulfide ores can enable many benefits, including the potential reduction of environmental impacts. The cells involved in bioleaching tend to have limited halotolerance, and increased halotolerance could enable several benefits, including a reduction in the need for the use of freshwater resources. We show that the genetic modification of A. ferrooxidans for the overproduction of glutathione is a promising strategy to enable cells to resist the oxidative stress that can occur during growth in the presence of salt.


Subject(s)
Acidithiobacillus/genetics , Acidithiobacillus/metabolism , Glutathione Synthase/genetics , Iron/metabolism , Salt Tolerance/genetics , Acidithiobacillus/drug effects , Escherichia coli/genetics , Glutathione/biosynthesis , Hydrogen-Ion Concentration , Oxidation-Reduction , Reactive Oxygen Species/metabolism , Sodium Chloride/pharmacology
9.
Nano Lett ; 21(13): 5896-5904, 2021 Jul 14.
Article in English | MEDLINE | ID: mdl-34197125

ABSTRACT

Thick electrodes, although promising toward high-energy battery systems, suffer from restricted lithium-ion transport kinetics due to prolonged diffusion lengths and tortuous transport pathways. Despite the emerging low-tortuosity designs, capacity retention under higher current densities is still limited. Herein, we employ a modified ice-templating method to fabricate low-tortuosity porous electrodes with tunable wall thickness and channel width and systematically investigate the critical impacts of the fine structural parameters on the thick electrode electrochemistry. While the porous electrodes with thick walls show diminished capability under a C-rate larger than 1.5 C, those with thinner walls could maintain ∼70% capacity under 2.5 C. The superior capacity retention is ascribed to the fast diffusion into the thin lamellar walls compared with their thicker counterparts. This study provides deeper insights into structure-affected electrochemistry and opens up new perspective of 3D porous architectural designs for high-energy and high-power electrodes.

10.
Biotechnol Bioeng ; 118(8): 3225-3238, 2021 08.
Article in English | MEDLINE | ID: mdl-34086346

ABSTRACT

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that is commonly reported to exhibit diauxic population growth behavior where ferrous iron is oxidized before elemental sulfur when both are available, despite the higher energy content of sulfur. We have discovered sulfur dispersion formulations that enables sulfur oxidation before ferrous iron oxidation. The oxidation of dispersed sulfur can lower the culture pH within days below the range where aerobic ferrous iron oxidation can occur. Thus, ferric iron reduction can be observed quickly which had previously been reported over extended incubation periods with untreated sulfur. Therefore, we demonstrate that this substrate utilization pattern is strongly dependent on the cell loading in relation to sulfur concentration, sulfur surface hydrophobicity, and the pH of the culture. Our dispersed sulfur formulation, lig-sulfur, can be used to support the rapid antibiotic selection of plasmid-transformed cells, which is not possible in liquid cultures where ferrous iron is the main source of energy for these acidophiles. Furthermore, we find that media containing lig-sulfur supports higher production of green fluorescent protein compared to media containing ferrous iron. The use of dispersed sulfur is a valuable new tool for the development of engineered A. ferrooxidans strains and it provides a new method to control iron and sulfur oxidation behaviors.


Subject(s)
Acidithiobacillus/growth & development , Culture Media/chemistry , Iron/metabolism , Sulfur/metabolism , Oxidation-Reduction
11.
J Manuf Process ; 66: 211-219, 2021 Jun.
Article in English | MEDLINE | ID: mdl-34012359

ABSTRACT

Fully metallic micrometer-scale 3D architectures can be fabricated via a hybrid additive methodology combining multi-photon lithography with electrochemical deposition of metals. The methodology - referred to as two-photon templated electrodeposition (2PTE) - has significant design freedom that enables the creation of complicated, traditionally difficult-to-make, high aspect ratio metallic structures such as microneedles. These complicated geometries, combined with their fully metallic nature, can enable precision surgical applications such as inner ear drug delivery or fluid sampling. However, the process involves electrochemical deposition of metals into complicated 3D lithography patterns thicker than 500 µm. This causes potential and chemical gradients to develop within the 3D template, creating limitations to what can be designed. These limitations can be explored, understood, and overcome via numerical modeling. Herein we introduce a numerical model as a design tool that can predict growth for manufacturing complicated 3D metallic geometries. The model is successful in predicting the geometric result of 2PTE, and enables extraction of insights about geometric constraints through exploration of its mechanics.

12.
Adv Mater ; 33(26): e2101275, 2021 Jul.
Article in English | MEDLINE | ID: mdl-34028911

ABSTRACT

The ever-growing needs for renewable energy demand the pursuit of batteries with higher energy/power output. A thick electrode design is considered as a promising solution for high-energy batteries due to the minimized inactive material ratio at the device level. Most of the current research focuses on pushing the electrode thickness to a maximum limit; however, very few of them thoroughly analyze the effect of electrode thickness on cell-level energy densities as well as the balance between energy and power density. Here, a realistic assessment of the combined effect of electrode thickness with other key design parameters is provided, such as active material fraction and electrode porosity, which affect the cell-level energy/power densities of lithium-LiNi0.6 Mn0.2 Co0.2 O2 (Li-NMC622) and lithium-sulfur (Li-S) cells as two model battery systems, is provided. Based on the state-of-the-art lithium batteries, key research targets are quantified to achieve 500 Wh kg-1 /800 Wh L-1 cell-level energy densities and strategies are elaborated to simultaneously enhance energy/power output. Furthermore, the remaining challenges are highlighted toward realizing scalable high-energy/power energy-storage systems.

13.
Drug Deliv Transl Res ; 11(1): 214-226, 2021 02.
Article in English | MEDLINE | ID: mdl-32488817

ABSTRACT

Drug delivery into the inner ear is a significant challenge due to its inaccessibility as a fluid-filled cavity within the temporal bone of the skull. The round window membrane (RWM) is the only delivery portal from the middle ear to the inner ear that does not require perforation of bone. Recent advances in microneedle fabrication enable the RWM to be perforated safely with polymeric microneedles as a means to enhance the rate of drug delivery from the middle ear to the inner ear. However, the polymeric material is not biocompatible and also lacks the strength of other materials. Herein we describe the design and development of gold-coated metallic microneedles suitable for RWM perforation. When developing microneedle technology for drug delivery, we considered three important general attributes: (1) high strength and ductility material, (2) high accuracy and precision of fabrication, and (3) broad design freedom. We developed a hybrid additive manufacturing method using two-photon lithography and electrochemical deposition to fabricate ultra-sharp gold-coated copper microneedles with these attributes. We refer to the microneedle fabrication methodology as two-photon templated electrodeposition (2PTE). We demonstrate the use of these microneedles by inducing a perforation with a minimal degree of trauma in a guinea pig RWM while the microneedle itself remains undamaged. Thus, this microneedle has the potential literally of opening the RWM for enhanced drug delivery into the inner ear. Finally, the 2PTE methodology can be applied to many different classes of microneedles for other drug delivery purposes as well the fabrication of small scale structures and devices for non-medical applications. Graphical Abstract Fully metallic ultra-sharp microneedle mounted at end of a 24-gauge stainless steel blunt syringe needle tip: (left) Size of microneedle shown relative to date stamp on U.S. one-cent coin; (right) Perforation through guinea pig round window membrane introduced with microneedle.


Subject(s)
Ear, Inner , Pharmaceutical Preparations , Animals , Drug Delivery Systems , Guinea Pigs , Needles , Round Window, Ear
14.
Phys Chem Chem Phys ; 23(1): 139-150, 2021 Jan 06.
Article in English | MEDLINE | ID: mdl-33025989

ABSTRACT

The phase distribution of lithiated LVO in thick (∼500 µm) porous electrodes (TPEs) designed to facilitate both ion and electron transport was determined using synchrotron-based operando energy dispersive X-ray diffraction (EDXRD). Probing 3 positions in the TPE while cycling at a 1C rate revealed a homogeneous phase transition across the thickness of the electrode at the 1st and 95th cycles. Continuum modelling indicated uniform lithiation across the TPE in agreement with the EDXRD results and ascribed decreasing accessible active material to be the cause of loss in delivered capacity between the 1st and 95th cycles. The model was supported by the observation of significant particle fracture by SEM consistent with loss of electrical contact. Overall, the combination of operando EDXRD, continuum modeling, and ex situ measurements enabled a deeper understanding of lithium vanadium oxide transport properties under high rate extended cycling within a thick highly porous electrode architecture.

15.
Biotechnol Bioeng ; 117(11): 3475-3485, 2020 11.
Article in English | MEDLINE | ID: mdl-32687219

ABSTRACT

Acidithiobacillus ferrooxidans cells can oxidize iron and sulfur and are key members of the microbial biomining communities that are exploited in the large-scale bioleaching of metal sulfide ores. Some minerals are recalcitrant to bioleaching due to the presence of other inhibitory materials in the ore bodies. Additives are intentionally included in processed metals to reduce environmental impacts and microbially influenced corrosion. We have previously reported a new aerobic corrosion mechanism where A. ferrooxidans cells combined with pyrite and chloride can oxidize low-grade stainless steel (SS304) with a thiosulfate-mediated mechanism. Here we explore process conditions and genetic engineering of the cells that enable corrosion of a higher grade steel (SS316). The addition of elemental sulfur and an increase in the cell loading resulted in a 74% increase in the corrosion of SS316 as compared to the initial sulfur- and cell-free control experiments containing only pyrite. The overexpression of the endogenous rus gene, which is involved in the cellular iron oxidation pathway, led to a further 85% increase in the corrosion of the steel in addition to the improvements made by changes to the process conditions. Thus, the modification of the culturing conditions and the use of rus-overexpressing cells led to a more than threefold increase in the corrosion of SS316 stainless steel, such that 15% of the metal coupons was dissolved in just 2 weeks. This study demonstrates how the engineering of cells and the optimization of their cultivation conditions can be used to discover conditions that lead to the corrosion of a complex metal target.


Subject(s)
Acidithiobacillus , Azurin , Stainless Steel , Acidithiobacillus/genetics , Acidithiobacillus/metabolism , Azurin/genetics , Azurin/metabolism , Corrosion , Iron/metabolism , Oxidation-Reduction , Sulfides/metabolism
16.
Protein Sci ; 29(9): 1890-1901, 2020 09.
Article in English | MEDLINE | ID: mdl-32654247

ABSTRACT

The COVID-2019 pandemic is the most severe acute public health threat of the twenty-first century. To properly address this crisis with both robust testing and novel treatments, we require a deep understanding of the life cycle of the causative agent, the SARS-CoV-2 coronavirus. Here, we examine the architecture and self-assembly properties of the SARS-CoV-2 nucleocapsid protein, which packages viral RNA into new virions. We determined a 1.4 Å resolution crystal structure of this protein's N2b domain, revealing a compact, intertwined dimer similar to that of related coronaviruses including SARS-CoV. While the N2b domain forms a dimer in solution, addition of the C-terminal spacer B/N3 domain mediates formation of a homotetramer. Using hydrogen-deuterium exchange mass spectrometry, we find evidence that at least part of this putatively disordered domain is structured, potentially forming an α-helix that self-associates and cooperates with the N2b domain to mediate tetramer formation. Finally, we map the locations of amino acid substitutions in the N protein from over 38,000 SARS-CoV-2 genome sequences. We find that these substitutions are strongly clustered in the protein's N2a linker domain, and that substitutions within the N1b and N2b domains cluster away from their functional RNA binding and dimerization interfaces. Overall, this work reveals the architecture and self-assembly properties of a key protein in the SARS-CoV-2 life cycle, with implications for both drug design and antibody-based testing.


Subject(s)
Coronavirus Nucleocapsid Proteins/biosynthesis , Coronavirus Nucleocapsid Proteins/metabolism , SARS-CoV-2/metabolism , Amino Acid Sequence , Amino Acid Substitution/genetics , COVID-19 , Crystallography, X-Ray , Genome, Viral/genetics , Humans , Protein Domains , RNA, Viral/metabolism
17.
bioRxiv ; 2020 Jun 08.
Article in English | MEDLINE | ID: mdl-32511359

ABSTRACT

The COVID-2019 pandemic is the most severe acute public health threat of the twenty-first century. To properly address this crisis with both robust testing and novel treatments, we require a deep understanding of the life cycle of the causative agent, the SARS-CoV-2 coronavirus. Here, we examine the architecture and self-assembly properties of the SARS-CoV-2 nucleocapsid protein, which packages viral RNA into new virions. We determined a 1.4 Å resolution crystal structure of this protein's N2b domain, revealing a compact, intertwined dimer similar to that of related coronaviruses including SARS-CoV. While the N2b domain forms a dimer in solution, addition of the C-terminal spacer B/N3 domain mediates formation of a homotetramer. Using hydrogen-deuterium exchange mass spectrometry, we find evidence that at least part of this putatively disordered domain is structured, potentially forming an α-helix that self-associates and cooperates with the N2b domain to mediate tetramer formation. Finally, we map the locations of amino acid substitutions in the N protein from over 38,000 SARS-CoV-2 genome sequences. We find that these substitutions are strongly clustered in the protein's N2a linker domain, and that substitutions within the N1b and N2b domains cluster away from their functional RNA binding and dimerization interfaces. Overall, this work reveals the architecture and self-assembly properties of a key protein in the SARS-CoV-2 life cycle, with implications for both drug design and antibody-based testing.

18.
J Biol Chem ; 295(9): 2804-2821, 2020 02 28.
Article in English | MEDLINE | ID: mdl-31964714

ABSTRACT

Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate-protein interactions. The identification of membrane heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-type lectin 14a (CLEC14A), a member of the C-type lectin family that modulates angiogenesis. We found that the C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.


Subject(s)
Cell Adhesion Molecules/metabolism , Endothelium/chemistry , Heparitin Sulfate/metabolism , Lectins, C-Type/metabolism , Membrane Proteins/metabolism , Proteomics/methods , Animals , Binding Sites , Cells, Cultured , Endothelium/cytology , Humans , Mice , Protein Binding , U937 Cells
19.
Appl Environ Microbiol ; 85(21)2019 11 01.
Article in English | MEDLINE | ID: mdl-31444204

ABSTRACT

Microbially influenced corrosion (MIC) results in significant damage to metallic materials in many industries. Anaerobic sulfate-reducing bacteria (SRB) have been well studied for their involvement in these processes. Highly corrosive environments are also found in pulp and paper processing, where chloride and thiosulfate lead to the corrosion of stainless steels. Acidithiobacillus ferrooxidans is a critically important chemolithotrophic acidophile exploited in metal biomining operations, and there is interest in using A. ferrooxidans cells for emerging processes such as electronic waste recycling. We explored conditions under which A. ferrooxidans could enable the corrosion of stainless steel. Acidic medium with iron, chloride, low sulfate, and pyrite supplementation created an environment where unstable thiosulfate was continuously generated. When combined with the chloride, acid, and iron, the thiosulfate enabled substantial corrosion of stainless steel (SS304) coupons (mass loss, 5.4 ± 1.1 mg/cm2 over 13 days), which is an order of magnitude higher than what has been reported for SRB. There results were verified in an abiotic flow reactor, and the importance of mixing was also demonstrated. Overall, these results indicate that A. ferrooxidans and related pyrite-oxidizing bacteria could produce aggressive MIC conditions in certain environmental milieus.IMPORTANCE MIC of industrial equipment, gas pipelines, and military material leads to billions of dollars in damage annually. Thus, there is a clear need to better understand MIC processes and chemistries as efforts are made to ameliorate these effects. Additionally, A. ferrooxidans is a valuable acidophile with high metal tolerance which can continuously generate ferric iron, making it critical to copper and other biomining operations as well as a potential biocatalyst for electronic waste recycling. New MIC mechanisms may expand the utility of these cells in future metal resource recovery operations.


Subject(s)
Acidithiobacillus/metabolism , Iron/chemistry , Stainless Steel/chemistry , Sulfates/chemistry , Thiosulfates/chemistry , Alloys , Chemoautotrophic Growth , Copper , Corrosion , Electrons , Industrial Microbiology , Mining , Oxidants , Oxidation-Reduction , Sulfides , Surface Properties
20.
Acad Med ; 94(12): 1910-1915, 2019 12.
Article in English | MEDLINE | ID: mdl-31335816

ABSTRACT

PROBLEM: Identifying and processing medical errors are overlooked components of undergraduate medical education. Organizations and leaders advocate teaching medical students about patient safety and medical error, yet few feasible examples demonstrate how this teaching should occur. To provide students with familiarity in identifying, reporting, and analyzing medical errors, the authors developed the interactive patient safety reporting curriculum (PSRC), requiring clinical students to engage intellectually and emotionally with personally experienced events in which the safety of one of their patients was compromised. APPROACH: In 2015, the authors incorporated the PSRC into the third-year internal medicine clerkship. Students completed a structured written report, analyzing a patient safety incident they experienced. The report focused on severity of outcome, root cause(s) analysis, system-based prevention, and personal reflection. The report was bookended by 2 interactive, case-based sessions led by faculty with expertise in patient safety, quality improvement, and medical errors. OUTCOMES: Students accurately analyzed the severity of the outcome, and their reports directly led to 2 formal root cause analyses and 4 system-based improvements. NEXT STEPS: The time- and resource-efficient PSRC allows students to apply patient safety knowledge to a medical error they experienced in a way that can directly affect care delivery. This model-interactive learning sessions coupled with engaging in a personally experienced case-can be implemented in various settings. Educators seeking to use student-experienced events for learning should not discount the emotional effects of those events on medical students.


Subject(s)
Clinical Clerkship/methods , Curriculum , Education, Medical, Undergraduate/methods , Medical Errors , Patient Safety , Risk Management/methods , Students, Medical/psychology , Comprehension , Humans , Internal Medicine/education , Medical Errors/prevention & control , Medical Errors/psychology , Program Development , Program Evaluation , United States
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