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Protein self-assembly, guided by the interplay of sequence- and environment-dependent liquid-liquid phase separation (LLPS), constitutes a fundamental process in the assembly of numerous intrinsically disordered proteins. Heuristic examination of these proteins has underscored the role of tyrosine residues, evident in their conservation and pivotal involvement in initiating LLPS and subsequent liquid-solid phase transitions (LSPT). The development of tyrosine-templated constructs, designed to mimic their natural counterparts, emerges as a promising strategy for creating adaptive, self-assembling systems with diverse applications. This review explores the central role of tyrosine in orchestrating protein self-assembly, delving into key interactions and examining its potential in innovative applications, including responsive biomaterials and bioengineering., Competing Interests: Declaration of interests The authors have no interests to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Longitudinal studies that continuously generate data enable the capture of temporal variations in experimentally observed parameters, facilitating the interpretation of results in a time-aware manner. We propose IL-VIS (incrementally learned visualizer), a new machine learning pipeline that incrementally learns and visualizes a progression trajectory representing the longitudinal changes in longitudinal studies. At each sampling time point in an experiment, IL-VIS generates a snapshot of the longitudinal process on the data observed thus far, a new feature that is beyond the reach of classical static models. We first verify the utility and correctness of IL-VIS using simulated data, for which the true progression trajectories are known. We find that it accurately captures and visualizes the trends and (dis)similarities between high-dimensional progression trajectories. We then apply IL-VIS to longitudinal multi-electrode array data from brain cortical organoids when exposed to different levels of quinolinic acid, a metabolite contributing to many neuroinflammatory diseases including Alzheimer's disease, and its blocking antibody. We uncover valuable insights into the organoids' electrophysiological maturation and response patterns over time under these conditions., (© 2024. The Author(s).)

Organohalides are recalcitrant, toxic environmental pollutants. Reductive dehalogenase enzymes (RDases) found in organohalide respiring bacteria (OHRB) utilise organohalides as electron acceptors for cellular energy and growth, producing lesser-halogenated compounds. Consequently, microbial reductive dehalogenation via organohalide respiration represents a promising solution for clean-up of organohalide pollutants. Dehalobacter sp. UNSWDHB is an OHRB capable of respiring highly toxic chloroform (CF) and converting it to dichloromethane (DCM). TmrA has been identified as an RDase responsible for this conversion and different strategies for generation of functional recombinant TmrA is the focus of this article. In this study, TmrA was recovered from inclusion bodies expressed in E. coli and refolded in the presence of FeCl 3 , Na 2 S and cobalamin to yield functional enzyme. TmrA has been previously expressed in a soluble and functional form in the corrinoid-producing Bacillus megaterium. Using a fractional experimental design for cultivation and induction combined with purification under anaerobic conditions resulted in substantially higher activity of recombinant and native TmrA than previously reported. TmrA was then expressed in a soluble and active form in Shimwellia blattae. Co-expression with two different putative chaperone proteins from the original host did not increase the level of soluble expression in S. blattae, however activity assays showed that removing the TAT signal from TmrA increases the dechlorination activity compared to when the TAT signal is present. Finally, TmrA was successfully expressed in a soluble and active form in the H 2 -oxidizing C. necator H16, a novel host for the expression of RDases., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Several specific syndromes within the RASopathies spectrum lead to an increased risk of seizures up to developing refractory epileptic encephalopathy. Management remains symptomatic., Methods: Here we report two patients treated with trametinib, a MEK1-2 inhibitor, as a precision strategy for drug-resistant epilepsy. Patient 1 is a six-year-old girl with cardiofaciocutaneous syndrome (BRAF p.F595L, germline mutation), and Patient 2 is a 14-month-old boy with Schimmelpenning syndrome (KRAS p.G12D, postzygotic somatic mutation). Trametinib was initiated at a dosage of 0.025 mg/kg/day., Results: Patient 1 had multiple seizures per day, multifocal motor to bilateral tonic-clonic. Electroencephalography (EEG) showed a dramatic reduction in EEG discharges three months after trametinib onset, while a marked clinical improvement occurred after about five months, at the same dosage, and the girl is currently seizure-free for more than six months. Patient 2 had left cerebral hemiatrophy leading to right focal motor seizures, multiple per week to multiple per day, since the age of three months. On trametinib, he experienced an early benefit, remaining seizure-free for more than three months. However, after six months we observed recurrence of seizures. After 22 months of treatment, trametinib was discontinued because of a suspected drug-induced inflammatory colitis. After discontinuation, we observed a significant clinical and EEG "rebound effect.", Conclusions: We provide proof of concept that MEK inhibition is a promising approach for the treatment of patients with refractory epilepsy with selected germline and mosaic RASopathies. Future trials are encouraged to better investigate their potentials and limitations., Competing Interests: Declaration of competing interest S.P. receives research support from Novartis-trametinib for a clinical trial. In kind drug support for the TRAM-01 study NCT03363217. G.D’O., M.-A.D., A.L., P.S., C.M., and G.A. declare no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Introduction: There are limited options for pain and distress management in children undergoing minor procedures, without the burden of an intravenous line insertion. Prior to this study, we conducted a dose-escalation study and identified 6 mg/kg as a potentially optimal initial dose of intranasal ketamine., Objective: To assess the efficacy and safety of intranasal ketamine at a dose of 6 mg/kg for procedural sedation to repair lacerations with sutures in children in the emergency department., Methods: We conducted a single-arm, open-label multicenter clinical trial for intranasal ketamine for laceration repair with sutures in children aged 1 to 12 years. A convenience sample of 30 patients received 6 mg/kg of intranasal ketamine for their procedural sedation. The primary outcome was the proportion (95% CI) of patients who achieved an effective procedural sedation., Results: We recruited 30 patients from April 2018 to December 2019 in two pediatric emergency departments in Canada. Lacerations repaired were mostly facial in 21(70%) patients and longer than 2 cm in 20 (67%) patients. Sedation was effective in 18/30 (60% [95% CI 45, 80]) children and was suboptimal in 5 (17%) patients but procedure was completed in them with minimal difficulties. Sedation was poor in the remaining 7 (23%) patients, with 3 (10%) of them required additional sedative agents. No serious adverse events were reported., Conclusions: Using a single dose of 6 mg/kg of intranasal Ketamine for laceration repair led to successful sedation in 60% of patients according to our a priori definition. An additional 17% of patients were considered suboptimal, but their procedure was still completed with minimal difficulty., Clinical Trial Registration: ClinicalTrials.gov (NCT03053947)., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest relevant to this article to disclose., (Copyright © 2023. Published by Elsevier Inc.)

Background: DNA methylation is a critical molecular mark involved in cellular differentiation and cell-specific processes. Single-cell whole genome DNA methylation profiling methods hold great potential to resolve the DNA methylation profiles of individual cell-types. Here we present a method that couples single-cell combinatorial indexing (sci) with enzymatic conversion (sciEM) of unmethylated cytosines., Results: The sciEM method facilitates DNA methylation profiling of single-cells that is highly correlated with single-cell bisulfite-based workflows (r 2  > 0.99) whilst improving sequencing alignment rates, reducing adapter contamination and over-estimation of DNA methylation levels (CpG and non-CpG). As proof-of-concept we perform sciEM analysis of the temporal lobe, motor cortex, hippocampus and cerebellum of the human brain to resolve single-cell DNA methylation of all major cell-types., Conclusion: To our knowledge sciEM represents the first non-bisulfite single-cell DNA methylation sequencing approach with single-base resolution., (© 2022. The Author(s).)

Laboratory models of the tumor microenvironment require control of mechanical and biochemical properties to ensure accurate mimicry of patient disease. In contrast to pure natural or synthetic materials, hybrid approaches that pair recombinant protein fragments with synthetic scaffolding show many advantages. Here we demonstrate production of a recombinant bacterial collagen-like protein (CLP) for thiol-ene pairing to norbornene functionalized hyaluronic acid (NorHA). The resultant hydrogel material shows an adjustable modulus with evidence for strain-stiffening behavior that resembles natural tumor matrices. Cysteine terminated peptide binding motifs are incorporated to adjust the cell-adhesion points. The modular hybrid gel shows good biocompatibility and was demonstrated to control cell adhesion, proliferation, and the invasive properties of MCF7 and MD-MBA-231 breast adenocarcinoma cells. The ease in which multiple structural and bioactive components can be integrated provides a robust framework to form models of the tumor microenvironment for fundamental studies and drug development.

Lanthanide-based beta-tricalcium phosphate (β-TCP) upconversion nanoparticles are exploited as a non-viral vector for imaging guided-gene therapy by virtue of their unique optical properties and multi-modality imaging ability, high transfection efficiency, high biocompatibility, dispersibility, simplicity of synthesis and surface modification. Ytterbium and thulium-doped β-TCP nanoparticles (βTCPYbTm) are synthesized via co-precipitation method, coated with polyethylenimine (PEI) and functionalized with a nuclear-targeting peptide (TAT). Further, in vitro studies revealed that the nanotheranostic carriers are able to transfect cells with the plasmid eGFP at a high efficiency, with approximately 60% of total cells producing the fluorescent green protein. The optimized protocol developed comprises the most efficient βTCPYbTm/PEI configuration, the amount and the order of assembly of βTCPYbTm:PEI, TAT, plasmid DNA and the culturing conditions. With having excellent dispersibility and high chemical affinity toward nucleic acid, calcium ions released from βTCPYbTm:PEI nanoparticles can participate in delivering nucleic acids and other therapeutic molecules, overcoming the nuclear barriers and improving the transfection efficacy. Equally important, the feasibility of the upconversion multifunctional nanovector to serve as an effective contrast agent for imaging modality, capable of converting low-energy light to higher-energy photons via a multi-photons mechanism, endowing greater unique luminescent properties, was successfully demonstrated., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Drug resistance among parasitic nematodes has resulted in an urgent need for the development of new therapies. However, the high re-discovery rate of anti-nematode compounds from terrestrial environments necessitates a new repository for future drug research. Marine epiphytes are hypothesised to produce nematicidal compounds as a defence against bacterivorous predators, thus representing a promising yet underexplored source for anti-nematode drug discovery. The marine epiphytic bacterium Pseudoalteromonas tunicata is known to produce several bioactive compounds. Screening heterologously expressed genomic libraries of P. tunicata against the nematode Caenorhabditis elegans , identified as an E. coli clone (HG8), shows fast-killing activity. Here we show that clone HG8 produces a novel nematode-killing protein-1 (Nkp-1) harbouring a predicted carbohydrate-binding domain with weak homology to known bacterial pore-forming toxins. We found bacteria expressing Nkp-1 were able to colonise the C. elegans intestine, with exposure to both live bacteria and protein extracts resulting in physical damage and necrosis, leading to nematode death within 24 h of exposure. Furthermore, this study revealed C. elegans dar (deformed anal region) and internal hatching may act as a nematode defence strategy against Nkp-1 toxicity. The characterisation of this novel protein and putative mode of action not only contributes to the development of novel anti-nematode applications in the future but reaffirms the potential of marine epiphytic bacteria as a new source of novel biomolecules.

Biopolymer-capped particles, sodium alginate-, gelatin- and reconstituted silk fibroin-capped nanosilver (AgNPs), were synthesized with an intention to study, simultaneously, their in vitro and in vivo haemocompatibility, one of the major safety factors in biomedical applications. Solid state characterization showed formation of spherical nanoparticles with 5 to 30 nm primary sizes (transmission electron microscopy) and X-ray photoelectron spectroscopy analysis of particles confirmed silver bonding with the biopolymer moieties. The degree of aggregation of the biopolymer-capped AgNPs in the synthesis medium (ultrapure water) is relatively low, with comparable hydrodynamic size with those of the control citrate-stabilized NPs, and remained relatively unchanged even after 6 weeks. The polymer-capped nanoparticles showed different degrees of aggregation in biologically relevant media - PBS (pH 7.4) and 2% human blood plasma - with citrate- (control) and alginate-capped particles showing the highest aggregation, while gelatin- and silk fibroin-capped particles revealed better stability and less aggregation in these media. In vitro cytotoxicity studies revealed that the polymer-capped particles exhibited both concentration and (hydrodynamic) size-dependent haemolytic activity, the extent of which was higher (up to 100% in some cases) in collected whole blood samples of healthy human volunteers when compared to that in the washed erythrocytes. This difference is thought to result from the detected protein corona formation on the nanoparticle surface in the whole blood system, which was associated with reduced particle aggregation, causing more severe cytotoxic effects. At the tested particle concentration range in vitro , we observed a negligible haemolysis effect in vivo (Balb/c mice). Polymer-capped particles did accumulate in organs, with the highest levels detected in the liver (up to 422 μg per g tissue), yet no adverse behavioural effects were observed in the mice during the duration of the nanoparticle exposure., Competing Interests: The authors have no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Objective: To describe the use of prophylactic inhaled antibiotics in children with a tracheostomy and assess if its use is associated with a reduction in exposition to broad-spectrum antibiotics and a lower risk of acquired respiratory tract infections. Methods: A case series study was performed in a tertiary care university affiliated hospital. All consecutive children (<18 years old) with a tracheostomy, hospitalized between January 2004 and November 2016, and treated with prophylactic inhaled antibiotics were identified. We analyzed the 3 month- period before and after initiation of prophylactic inhaled antibiotics and described exposure to broad spectrum antibiotics, the number of respiratory tract infections and the associated adverse events. Results: Six children (median age: 11 months, range: 8-100) were included. One received colimycin, 3 received tobramycin and 2 were treated with both antibiotics in alternance. The median duration of treatment was 74 days (22-173) with one patient still being treated at the end of the study. Patients were exposed to systemic antibiotics for 18 days (2-49) in the 3 months preceding the treatment vs. 2 days (0-15) in the 3 months following the treatment initiation ( p = 0.115). The number of respiratory tract infections went from median of 2 (0-3) to 1 (0-1) during the same periods ( p = 0.07). Adverse events most commonly reported were cough ( n = 2) and increased respiratory secretions post-inhalation ( n = 4). Only one new bacterial resistance was observed. Conclusions: This series of consecutive cases underlines the need for future studies evaluating the potential benefit of prophylactic inhaled antibiotics in children with a tracheostomy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jutras, Autmizguine, Chomton, Marquis, Nguyen, Roumeliotis and Emeriaud.)

A hydrocyclone is a macroscale separation device employed in various industries, with many advantages, including high-throughput and low operational costs. Translating these advantages to microscale has been a challenge due to the microscale fabrication limitations that can be surmounted using 3D printing technology. Additionally, it is difficult to simulate the performance of real 3D-printed micro-hydrocyclones because of turbulent eddies and the deviations from the design due to printing resolution. To address these issues, we propose a new experimental method for the direct observation of particle motion in 3D printed micro-hydrocyclones. To do so, wax 3D printing and soft lithography were used in combination to construct a transparent micro-hydrocyclone in a single block of polydimethylsiloxane. A high-speed camera and fluorescent particles were employed to obtain clear in situ images and to confirm the presence of the vortex core. To showcase the use of this method, we demonstrate that a well-designed device can achieve a 95% separation efficiency for a sample containing a mixture of (desired) stem cells and (undesired) microcarriers. Overall, we hope that the proposed method for the direct visualization of particle trajectories in micro-hydrocyclones will serve as a tool, which can be leveraged to accelerate the development of micro-hydrocyclones for biomedical applications., (© 2020 Author(s).)

Growth Differentiation Factor-15 (GDF15) is a divergent TGF-beta superfamily cytokine that is overexpressed by most cancers and is induced by anticancer therapy. Transgenic and induced animal models suggest that it protects from cancer development but the mechanisms are uncertain. We investigated the role of immunity in GDF15 induced reduction in prostate cancer (PCa) growth. The C57BL/6 transgenic TRAMP prostate cancer prone mice were bred with mice that were immunodeficient and/or systemically overexpressed GDF15. We developed a novel orthotopic TRAMP PCa model in which primary TRAMP tumor cells were implanted into prostates of mice to reduce the study time. These mice were administered recombinant mouse GDF15, antibody to CD8, PD1 or their respective controls. We found that GDF15 induced protection from tumor growth was reversed by lack of adaptive immunity. Flow cytometric evaluation of lymphocytes within these orthotopic tumors showed that GDF15 overexpression was associated with increased CD8 T cell numbers and an increased number and proportion of recently activated CD8+CD11c+ T cells and a reduced proportion of "exhausted" CD8+PD1+ T cells. Further, depletion of CD8 T cells in tumor bearing mice abolished the GDF15 induced protection from tumor growth. Infusion of GDF15 into mice bearing orthotopic TRAMP tumor, substantially reduced tumor growth that was further reduced by concurrent PD1 antibody administration. GDF15 overexpression or recombinant protein protects from TRAMP tumor growth by modulating CD8 T cell mediated antitumor immunity and augments the positive effects of anti-PD1 blockers., Competing Interests: DAB and SNB are named inventors on patents owned by St Vincent’s Hospital that pertain to the clinical use of a MIC-1/GDF15 diagnostic assay and modulatory therapy. DAB and SNB declare no competing non-financial interests. YH, VWWT, RM, HPZ, KMLN, HL and CPM declare no competing financial and/or nonfinancial interests. St Vincent’s Hospital agrees to make freely available any materials and information described in this publication that may be reasonably requested for the purpose of academic, non-commercial research. Due to the proprietary nature of the materials, the parties will need to enter into a material transfer agreement. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

The work describes the interactions of nanosilver (NAg) with bacterial cell envelope components at a molecular level and how this associates with the reactive oxygen species (ROS)-mediated toxicity of the nanoparticle. Major structural changes were detected in cell envelope biomolecules as a result of damages in functional moieties, such as the saccharides, amides, and phosphodiesters. NAg exposure disintegrates the glycan backbone in the major cell wall component peptidoglycan, causes complete breakdown of lipoteichoic acid, and disrupts the phosphate-amine and fatty acid groups in phosphatidylethanolamine, a membrane phospholipid. Consistent with the oxidative attacks, we propose that the observed cell envelope damages are inflicted, at least in part, by the reactive oxygen radicals being generated by the nanoparticle during its leaching process, abiotically, without cells. The cell envelope targeting, especially those on the inner membrane phospholipid, is likely to then trigger the rapid generation of lethal levels of cellular superoxide (O 2 •- ) and hydroxyl (OH • ) radicals herein seen with a model bacterium. The present study provides a better understanding of the antibacterial mechanisms of NAg, whereby ROS generation could be both the cause and consequence of the toxicity, associated with the initial cell envelope targeting by the nanoparticle.

Background: Elevated circulating levels of the divergent transforming growth factor-beta (TGFb) family cytokine, growth differentiation factor 15 (GDF15), acting through its CNS receptor, glial-derived neurotrophic factor receptor alpha-like (GFRAL), can cause anorexia and weight loss leading to anorexia/cachexia syndrome of cancer and other diseases. Preclinical studies suggest that administration of drugs based on recombinant GDF15 might be used to treat severe obesity. However, the role of the GDF15-GFRAL pathway in the physiological regulation of body weight and metabolism is unclear. The critical site of action of GFRAL in the CNS has also not been proven beyond doubt. To investigate these two aspects, we have inhibited the actions of GDF15 in mice started on high-fat diet (HFD)., Methods: The actions of GDF15 were inhibited using two methods: (1) Groups of 8 mice under HFD had their endogenous GDF15 neutralised by monoclonal antibody treatment, (2) Groups of 15 mice received AAV-shRNA to knockdown GFRAL at its hypothesised major sites of action, the hindbrain area postrema (AP) and the nucleus of the solitary tract (NTS). Metabolic measurements were determined during both experiments., Conclusions: Treating mice with monoclonal antibody to GDF15 shortly after commencing HFD results in more rapid gain of body weight, adiposity and hepatic lipid deposition than the control groups. This is accompanied by reduced glucose and insulin tolerance and greater expression of pro-inflammatory cytokines in adipose tissue. Localised AP and NTS shRNA-GFRAL knockdown in mice commencing HFD similarly caused an increase in body weight and adiposity. This effect was in proportion to the effectiveness of GFRAL knockdown, indicated by quantitative analysis of hindbrain GFRAL staining. We conclude that the GDF15-GFRAL axis plays an important role in resistance to obesity in HFD-fed mice and that the major site of action of GDF15 in the CNS is GFRAL-expressing neurons in the AP and NTS.

The purpose of the study was to evaluate the influence of establishing a protocol for the use of combined sodium benzoate and sodium phenylacetate (SBSP) (Ammonul®) to treat acute hyperammonemia. This was a retrospective, single-center study in a 24-bed medical and surgical pediatric intensive care unit (PICU) in a tertiary care teaching maternal-child hospital in Canada. Inclusion criteria were age < 18 years, PICU admission between 1 January 2000 and 30 June 2016, and SBSP treatment. An SBSP delivery protocol was implemented in our hospital on 30 August 2008 in order to improve management of acute hyperammonemia. Patients were assigned to one of the two groups, without or with protocol, depending on date of admission. SBSP was ordered 34 times during the study period, and 23 orders were considered for analysis (14 with and 9 without protocol). Patient characteristics were similar between groups. The median time from diagnosis to prescription was significantly shorter in the protocol group [40 min (21-82) vs 100 min (70-150), p = 0.03)] but the median time from diagnosis to administration of the treatment was equivalent [144 min (90-220) vs 195 (143-274), (p = 0.2)]. Other clinical outcomes did not differ. This study is the first to compare two SBSP delivery strategies in the treatment of acute hyperammonemia in this PICU setting. Implementation of a delivery protocol shortened the time from diagnosis of hyperammonemia to prescription of SBSP and helped us identify other parameters that can be improved to optimize treatment delivery., (© 2018 SSIEM.)

Nanosilver (Ag NPs) is currently one of the most commercialized antimicrobial nanoparticles with as yet, still unresolved cytotoxicity origins. To date, research efforts have mostly described the antimicrobial contribution from the leaching of soluble silver, while the undissolved solid Ag particulates are often considered as being microbiologically inert, serving only as source of the cytotoxic Ag ions. Here, we show the rapid stimulation of lethal cellular oxidative stress in bacteria by the presence of the undissolved Ag particulates. The cytotoxicity characteristics are distinct from those arising from the leached soluble Ag, the latter being locked in organic complexes. The work also highlights the unique oxidative stress-independent bacterial toxicity of silver salt. Taken together, the findings advocate that future enquiries on the antimicrobial potency and also importantly, the environmental and clinical impact of Ag NPs use, should pay attention to the potential bacterial toxicological responses to the undissolved Ag particulates, rather than just to the leaching of soluble silver. The findings also put into question the common use of silver salt as model material for evaluating bacterial toxicity of Ag NPs.

Reductive dehalogenases (RDases) are key enzymes involved in the respiratory process of anaerobic organohalide respiring bacteria (ORB). Heterologous expression of respiratory RDases is desirable for structural and functional studies; however, there are few reports of successful expression of these enzymes. Dehalobacter sp. strain UNSWDHB is an ORB, whose preferred electron acceptor is chloroform. This study describes efforts to express recombinant reductive dehalogenase (TmrA), derived from UNSW DHB, using the heterologous hosts Escherichia coli and Bacillus megaterium. Here, we report the recombinant expression of soluble and functional TmrA, using B. megaterium as an expression host under a xylose-inducible promoter. Successful incorporation of iron-sulfur clusters and a corrinoid cofactor was demonstrated using UV-vis spectroscopic analyses. In vitro dehalogenation of chloroform using purified recombinant TmrA was demonstrated. This is the first known report of heterologous expression and purification of a respiratory reductive dehalogenase from an obligate organohalide respiring bacterium.

We report the antimicrobial activity of bare and surface functionalized indium tin oxide (ITO) conjugated with T4 bacteriophage towards E. coli. A ∼ 10 3 -fold reduction (99.9%) in the bacterial concentration was achieved within 2 h exposure of E. coli to the bare as well as the amine, carboxylic and methyl functionalized ITO/T4 surfaces. Despite the known differences in bacteriophage loading of these ITO/T4 systems, the almost identical extent of antimicrobial activity of all of the ITO/T4 systems resulted from the release of a comparable amount of infective T4 from the systems. As anticipated, a single dose of immobilized bacteriophage was sufficient to eliminate further surge of bacterial population. Upon the 2 h eradication of the '1st batch' of E. coli population, all of the ITO/T4 systems, each system with 10 2 -fold more suspended bacteriophage (due to propagation of the phage at the expense of the '1st batch' E. coli death), reduced the '2nd batch' of E. coli concentration by ∼10 4 -fold in just 30 min, suggesting the potential of immobilized bacteriophage systems as solution to the issues of antimicrobial agent depletion. All of the ITO/T4 systems maintained their antimicrobial activity in the presence of model food components. The antimicrobial activity was however, affected by pH; at pH 5 whereby the bacteria's growth was physiologically inhibited, generally no reduction in E. coli concentration was detected. The present work provides an understanding of the mode of antimicrobial activity exhibited by an immobilized bacteriophage based substrate and demonstrates efficacy in the presence of food components., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A G protein-coupled receptor (GPCR) agonist protein, thaumatin, was site-specifically conjugated at the N- or C-terminus with a fluorophore for visualization of GPCR:agonist interactions. The N-terminus was specifically conjugated using a synthetic 2-pyridinecarboxyaldehyde reagent. The interaction profiles observed for N- and C-terminal conjugates were varied; N-terminal conjugates interacted very weakly with the GPCR of interest, whereas C-terminal conjugates bound to the receptor. These chemical biology tools allow interactions of therapeutic proteins:GPCR to be monitored and visualized. The methodology used for site-specific bioconjugation represents an advance in application of 2-pyridinecarboxyaldehydes for N-terminal specific bioconjugations.

The sweetest tasting molecule known is the protein thaumatin, first isolated from the katemfe fruit, Thaumatococcus daniellii. Thaumatin is used in the food and beverage industry as a low-calorie sugar substitute. Thaumatin interacts with taste receptors in the oral cavity eliciting a persistent sweet taste and a bitter, liquorice flavor. Recombinant thaumatin was expressed in Pichia pastoris and through a co-expression strategy with a molecular chaperone, yields of one engineered thaumatin variant increased by greater than two-fold. A detailed purification strategy for thaumatin is reported resulting in a homogenous sample recovered at a yield of 42%. The recombinant thaumatins were extensively characterised using size exclusion chromatography for homogeneity, reversed-phase HPLC for purity (99%), peptide digest LC-MS/MS for sequence determination, and circular dichroism and tryptophan fluorescence spectroscopies for conformational characterisation. These new thaumatin variants are amenable for bioconjugation, providing chemical biology tools for thaumatin:taste receptor interaction studies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We report herein the purification of a chloroform (CF)-reducing enzyme, TmrA, from the membrane fraction of a strict anaerobe Dehalobacter sp. strain UNSWDHB to apparent homogeneity with an approximate 23-fold increase in relative purity compared to crude lysate. The membrane fraction obtained by ultracentrifugation was solubilized in Triton X-100 in the presence of glycerol, followed by purification by anion exchange chromatography. The molecular mass of the purified TmrA was determined to be 44.5 kDa by SDS-PAGE and MALDI-TOF/TOF. The purified dehalogenase reductively dechlorinated CF to dichloromethane in vitro with reduced methyl viologen as the electron donor at a specific activity of (1.27 ± 0.04) × 10 3 units mg protein -1 . The optimum temperature and pH for the activity were 45°C and 7.2, respectively. The UV-visible spectrometric analysis indicated the presence of a corrinoid and two [4Fe-4S] clusters, predicted from the amino acid sequence. This is the first report of the production, purification and biochemical characterization of a CF reductive dehalogenase., (© 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

In this era of increasing antibiotic resistance, the use of alternative antimicrobials such as silver has become more widespread. Superior antimicrobial activity has been provided through fabrication of silver nanoparticles or nanosilver (NAg), which imparts cytotoxic actions distinct from those of bulk silver. In the wake of the recent discoveries of bacterial resistance to NAg and its rising incorporation in medical and consumer goods such as wound dressings and dietary supplements, we argue that there is an urgent need to monitor the prevalence and spread of NAg microbial resistance. In this Perspective, we describe how the use of NAg in commercially available products facilitates prolonged microorganism exposure to bioavailable silver, which underpins the development of resistance. Furthermore, we advocate for a judicial approach toward NAg use in order to preserve its efficacy and to avoid environmental disruption.

This work demonstrates the use of bacteriophage conjugated magnetic particles (Fe 3 O 4 ) for the rapid capturing and isolation of Escherichia coli. The investigation of T4 bacteriophage adsorption to silane functionalised Fe 3 O 4 with amine (NH 2 ), carboxylic (COOH) and methyl (CH 3 ) surface functional groups reveals the domination of net electrostatic and hydrophobic interactions in governing bacteriophage adsorption. The bare Fe 3 O 4 and Fe 3 O 4 -NH 2 with high T4 loading captured 3-fold more E. coli (∼70% capturing efficiency) compared to the low loading T4 on Fe 3 O 4 -COOH, suggesting the significance of T4 loading in E. coli capturing efficiency. Importantly, it is further revealed that E. coli capture is highly dependent on the incubation temperature and the presence of tryptone in the media. Effective E. coli capturing only occurs at 37°C in tryptone-containing media with the absence of either conditions resulted in poor bacteria capture. The incubation temperature dictates the capturing ability of Fe 3 O 4 /T4, whereby T4 and E. coli need to establish an irreversible binding that occurred at 37°C. The presence of tryptophan-rich tryptone in the suspending media was also critical, as shown by a 3-fold increase in E. coli capture efficiency of Fe 3 O 4 /T4 in tryptone-containing media compared to that in tryptone-free media. This highlights for the first time that successful bacteria capturing requires not only an optimum tailoring of the particle's surface physicochemical properties for favourable bacteriophage loading, but also an in-depth understanding of how factors, such as temperature and solution chemistry influence the subsequent bacteriophage-bacteria interactions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Organohalide respiring bacteria (ORB) are capable of utilising organohalides as electron acceptors for the generation of cellular energy and consequently play an important role in the turnover of natural and anthropogenically-derived organohalides. In this study, the response of a Dehalobacter sp. strain UNSWDHB to the addition of trichloromethane (TCM) after a 50 h period of its absence (suffocation) was evaluated from a transcriptomic and proteomic perspective. The up-regulation of TCM reductive dehalogenase genes (tmrABC) and their gene products (TmrABC) was confirmed at both transcriptional and proteomic levels. Other findings include the upregulation of various hydrogenases (membrane-associated Ni-Fe hydrogenase complexes and soluble Fe-Fe hydrogenases), formate dehydrogenases, complex I and a pyrophosphate-energized proton pump. The elevated expression of enzymes associated with carbon metabolism, including complete Wood Ljungdahl pathway, during TCM respiration raises interesting questions on possible fates of intracellular formate and its potential role in the physiology of this bacterium. Overall, the findings presented here provide a broader view on the bioenergetics and general physiology of Dehalobacter UNSWDHB cells actively respiring with TCM., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni-Fe] uptake hydrogenase (SH) produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.

The work investigates the influence of surface physicochemical properties of planar indium tin oxide (ITO) as a model substrate on T4 bacteriophage adsorption. A comparative T4 bacteriophage adsorption study shows a significant difference in bacteriophage adsorption observed on chemically modified planar ITO when compared to similarly modified particulate ITO, which infers that trends observed in virus-particle interaction studies are not necessarily transferrable to predict virus-planar surface adsorption behaviour. We also found that ITO surfaces modified with methyl groups, (resulting in increased surface roughness and hydrophobicity) remained capable of adsorbing T4 bacteriophage. The adsorption of T4 onto bare, amine and carboxylic functionalised planar ITO suggests the presence of a unique binding behaviour involving specific functional groups on planar ITO surface beyond the non-specific electrostatic interactions that dominate phage to particle interactions. The paper demonstrates the significance of physicochemical properties of surfaces on bacteriophage-surface interactions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The soluble hydrogenase (SH) from Ralstonia eutropha H16 is a promising candidate enzyme for H2-based biofuel application as it favours H2 oxidation and is relatively oxygen-tolerant. In this report, bioprocess development studies undertaken to produce and purify an active SH are described, based on the methods previously reported [1], [2], [3], [4]. Our modifications are: •Upstream method optimizations were undertaken on heterotrophic growth media and cell lysis involving ultrasonication.•Two anion exchangers (Q Sepharose and RESOURCE Q) and size exclusion chromatographic (Superdex 200) matrices were successfully employed for purification of a hexameric SH from R. eutropha.•The H2 oxidizing activity of the SH was demonstrated spectrophotometrically in solution and also immobilized on an EPG electrode using cyclic voltammetry.

Organohalides are recalcitrant pollutants that have been responsible for substantial contamination of soils and groundwater. Organohalide-respiring bacteria (ORB) provide a potential solution to remediate contaminated sites, through their ability to use organohalides as terminal electron acceptors to yield energy for growth (i.e., organohalide respiration). Ideally, this process results in non- or lesser-halogenated compounds that are mostly less toxic to the environment or more easily degraded. At the heart of these processes are reductive dehalogenases (RDases), which are membrane bound enzymes coupled with other components that facilitate dehalogenation of organohalides to generate cellular energy. This review focuses on RDases, concentrating on those which have been purified (partially or wholly) and functionally characterized. Further, the paper reviews the major bacteria involved in organohalide breakdown and the evidence for microbial evolution of RDases. Finally, the capacity for using ORB in a bioremediation and bioaugmentation capacity are discussed.

Halogenated organic compounds (organohalides) are globally prevalent, recalcitrant toxic, and carcinogenic environmental pollutants. Select microorganisms encode enzymes known as reductive dehalogenases (EC 1.97.1.8) that catalyze reductive dehalogenation reactions resulting in the generation of lesser-halogenated compounds that may be less toxic and more biodegradable. Recent breakthroughs in enzyme structure determination, elucidation of the mechanisms of reductive dehalogenation, and in heterologous expression of functional reductive dehalogenase enzymes have substantially increased our understanding of this fascinating class of enzymes. This knowledge has created opportunities for more versatile (in situ and ex situ) biologically-mediated organohalide destruction strategies., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The human and mouse antibody repertoires are formed by identical processes, but like all small animals, mice only have sufficient lymphocytes to express a small part of the potential antibody repertoire. In this study, we determined how the heavy chain repertoires of two mouse strains are generated. Analysis of IgM- and IgG-associated VDJ rearrangements generated by high-throughput sequencing confirmed the presence of 99 functional immunoglobulin heavy chain variable (IGHV) genes in the C57BL/6 genome, and inferred the presence of 164 IGHV genes in the BALB/c genome. Remarkably, only five IGHV sequences were common to both strains. Compared with humans, little N nucleotide addition was seen in the junctions of mouse VDJ genes. Germline human IgG-associated IGHV genes are rare, but many murine IgG-associated IGHV genes were unmutated. Together these results suggest that the expressed mouse repertoire is more germline-focused than the human repertoire. The apparently divergent germline repertoires of the mouse strains are discussed with reference to reports that inbred mouse strains carry blocks of genes derived from each of the three subspecies of the house mouse. We hypothesize that the germline genes of BALB/c and C57BL/6 mice may originally have evolved to generate distinct germline-focused antibody repertoires in the different mouse subspecies., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)

The TGF-b superfamily cytokine MIC-1/GDF15 circulates in the blood of healthy humans. Its levels rise substantially in cancer and other diseases and this may sometimes lead to development of an anorexia/cachexia syndrome. This is mediated by a direct action of MIC-1/GDF15 on feeding centres in the hypothalamus and brainstem. More recent studies in germline gene deleted mice also suggest that this cytokine may play a role in physiological regulation of energy homeostasis. To further characterize the role of MIC-1/GDF15 in physiological regulation of energy homeostasis in man, we have examined diurnal and food associated variation in serum levels and whether variation in circulating levels relate to BMI in human monozygotic twin pairs. We found that the within twin pair differences in serum MIC-1/GDF15 levels were significantly correlated with within twin pair differences in BMI, suggesting a role for MIC-1/GDF15 in the regulation of energy balance in man. MIC-1/GDF15 serum levels altered slightly in response to a meal, but comparison with variation its serum levels over a 24 hour period suggested that these changes are likely to be due to bimodal diurnal variation which can alter serum MIC-1/GDF15 levels by about plus or minus 10% from the mesor. The lack of a rapid and substantial postprandial increase in MIC-1/GDF15 serum levels suggests that MIC1/GDF15 is unlikely to act as a satiety factor. Taken together, our findings suggest that MIC-1/GDF15 may be a physiological regulator of energy homeostasis in man, most probably due to actions on long-term regulation of energy homeostasis.

Polymersomes provide a good platform for targeted drug delivery and the creation of complex (bio)catalytically active systems for research in synthetic biology. To realize these applications requires both spatial control over the encapsulation components in these polymersomes and a means to report where the components are in the polymersomes. To address these twin challenges, we synthesized the protein-polymer bioconjugate PNIPAM-b-amilFP497 composed of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and a green-fluorescent protein variant (amilFP497). Above 37 °C, this bioconjugate forms polymersomes that can (co-)encapsulate the fluorescent drug doxorubicin and the fluorescent light-harvesting protein phycoerythrin 545 (PE545). Using fluorescence lifetime imaging microscopy and Förster resonance energy transfer (FLIM-FRET), we can distinguish the co-encapsulated PE545 protein inside the polymersome membrane while doxorubicin is found both in the polymersome core and membrane., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Soluble hydrogenases (SH) are enzymes that catalyse the oxidation of molecular hydrogen. The SH enzyme from Cupriavidus necator H16 is relatively oxygen tolerant and makes an attractive target for potential application in biochemical hydrogen fuel cells. Expression of the enzyme can be mediated by derepression of the hox promoter system under heterotrophic conditions. However, the overall impact of hox derepression, from a transcriptomic perspective, has never been previously reported., Results: Derepression of hydrogenase gene expression upon fructose depletion was confirmed in replicate experiments. Using qRT-PCR, hoxF was 4.6-fold up-regulated, hypF2 was up-regulated in the cells grown 2.2-fold and the regulatory gene hoxA was up-regulated by a mean factor of 4.5. A full transcriptomic evaluation revealed a substantial shift in the global pattern of gene expression. In addition to up-regulation of genes associated with hydrogenase expression, significant changes were observed in genes associated with energy transduction, amino acid metabolism, transcription and translation (and regulation thereof), genes associated with cell stress, lipid and cell wall biogenesis and other functions, including cell motility., Conclusions: We report the first full transcriptome analysis of C. necator H16 grown heterotrophically on fructose and glycerol in diauxic batch culture, which permits expression of soluble hydrogenase under heterotrophic conditions. The data presented deepens our understanding of the changes in global gene expression patterns that occur during the switch to growth on glycerol and suggests that energy deficit is a key driver for induction of hydrogenase expression in this organism.

Despite the importance of protein dimers and dimerization in biology, the formation of protein dimers through synthetic covalent chemistry has not found widespread use. In the case of maleimide-cysteine-based dimerization of proteins, we show here that when the proteins have the same charge, dimerization appears to be inherently difficult with yields around 1% or less, regardless of the nature of the spacer used or whether homo- or heteroprotein dimers are targeted. In contrast, if the proteins have opposing (complementary) charges, the formation of heteroprotein dimers proceeds much more readily, and in the case of one high molecular weight (>80 kDa) synthetic dimer between cytochrome c and bovine serum albumin, a 30% yield of the purified, isolated dimer was achieved. This represents at least a 30-fold increase in yield for protein dimers formed from proteins with complementary charges, compared to when the proteins have the same charge, under otherwise similar conditions. These results illustrate the role of ionic supramolecular interactions in controlling the reactivity of proteins toward bis-functionalized spacers. The strategy here for effective synthetic dimerization of proteins could be very useful for developing novel approaches to study the important role of protein-protein interactions in chemical biology.

Macrophage inhibitory cytokine-1 (MIC-1/GDF15) modulates food intake and body weight under physiological and pathological conditions by acting on the hypothalamus and brainstem. When overexpressed in disease, such as in advanced cancer, elevated serum MIC-1/GDF15 levels lead to an anorexia/cachexia syndrome. To gain a better understanding of its actions in the brainstem we studied MIC-1/GDF15 induced neuronal activation identified by induction of Fos protein. Intraperitoneal injection of human MIC-1/GDF15 in mice activated brainstem neurons in the area postrema (AP) and the medial (m) portion of the nucleus of the solitary tract (NTS), which did not stain with tyrosine hydroxylase (TH). To determine the importance of these brainstem nuclei in the anorexigenic effect of MIC-1/GDF15, we ablated the AP alone or the AP and the NTS. The latter combined lesion completely reversed the anorexigenic effects of MIC-1/GDF15. Altogether, this study identified neurons in the AP and/or NTS, as being critical for the regulation of food intake and body weight by MIC-1/GDF15.

Upon contact with plasma or other protein-containing biological fluids, the surface of nanoparticles is immediately decorated with proteins forming a biologically active protein corona. The biological fates and functions of nanoparticles are determined by physiological responses toward these nanoparticle-protein corona complexes as the effective biological unit of nanoparticles. In this article, we review representative studies on the effects of particle physicochemical characteristics along with the protein profiles in the biological medium on the formation of protein corona and importantly, how the dynamic nature and protein fingerprints of the formed corona govern the biological responses toward nanoparticles. The biological effects arising from the presence of protein corona can be both beneficial and unfavourable to the biomedical applications of nanoparticles. The protein corona-cell interactions open up the feasibility of targeted delivery and cell-specific uptake of therapeutic nanoparticles and in other circumstances, engineering of nanoparticles as adjuvants for vaccine development as well as mitigation of the unintentional cytotoxic effects of nanoparticles. On the other hand, the protein corona-cell interactions could induce rapid clearance of nanoparticles from in vivo circulation as well as activating unwanted inflammatory responses. Taken together, the knowledge on the formation and biological effects of protein corona enables tailored tuning of the physicochemical characteristics of nanoparticles, unique to their intended biological activity.

The natural ability of Bacillus sp. to adapt to nanosilver cytotoxicity upon prolonged exposure is reported for the first time. The combined adaptive effects of nanosilver resistance and enhanced growth are induced under various intensities of nanosilver-stimulated cellular oxidative stress, ranging from only minimal cellular redox imbalance to the lethal levels of cellular ROS stimulation. An important implication of the present work is that such adaptive effects lead to the ultimate domination of nanosilver-resistant Bacillus sp. in the microbiota, to which nanosilver cytotoxicity is continuously applied., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) plays an essential role in the replication of HCV and is a key target for novel antiviral therapies. Several RdRp inhibitors are in clinical trials and have increased response rates when combined with current interferon-based therapies for genotype 1 (G1) HCV patients. These inhibitors, however, show poor efficacy against non-G1 genotypes, including G3a, which represents ~20% of HCV cases globally. Here, we used a commercially available fluorescent dye to characterize G3a HCV RdRp in vitro. RdRp activity was assessed via synthesis of double-stranded RNA from the single-stranded RNA poly(C) template. The assay was miniaturized to a 384-well microplate format and a pilot high-throughput screen was conducted using 10,208 "lead-like" compounds, randomly selected to identify inhibitors of HCV G3a RdRp. Of 150 compounds demonstrating greatest inhibition, 10 were confirmed using both fluorescent and radioactive assays. The top two inhibitors (HAC001 and HAC002) demonstrated specific activity, with an IC(50) of 12.7 µM and 1.0 µM, respectively. In conclusion, we describe simple, fluorescent-based high-throughput screening (HTS) for the identification of inhibitors of de novo RdRp activity, using HCV G3a RdRp as the target. The HTS system could be used against any positive-sense RNA virus that cannot be cultured.

The work investigates the eco-cytoxicity of submicron and nano TiO₂ and ZnO, arising from the unique interactions of freshwater microalga Chlamydomonas reinhardtii to soluble and undissolved components of the metal oxides. In a freshwater medium, submicron and nano TiO₂ exist as suspended aggregates with no-observable leaching. Submicron and nano ZnO undergo comparable concentration-dependent fractional leaching, and exist as dissolved zinc and aggregates of undissolved ZnO. Cellular internalisation of solid TiO₂ stimulates cellular ROS generation as an early stress response. The cellular redox imbalance was observed for both submicron and nano TiO₂ exposure, despite exhibiting benign effects on the alga proliferation (8-day EC50>100 mg TiO₂/L). Parallel exposure of C. reinhardtii to submicron and nano ZnO saw cellular uptake of both the leached zinc and solid ZnO and resulting in inhibition of the alga growth (8-day EC50≥0.01 mg ZnO/L). Despite the sensitivity, no zinc-induced cellular ROS generation was detected, even at 100 mg ZnO/L exposure. Taken together, the observations confront the generally accepted paradigm of cellular oxidative stress-mediated cytotoxicity of particles. The knowledge of speciation of particles and the corresponding stimulation of unique cellular responses and cytotoxicity is vital for assessment of the environmental implications of these materials., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Scleractinian corals occur in symbiosis with a range of organisms including the dinoflagellate alga, Symbiodinium, an association that is mutualistic. However, not all symbionts benefit the host. In particular, many organisms within the microbial mucus layer that covers the coral epithelium can cause disease and death. Other organisms in symbiosis with corals include the recently described Chromera velia, a photosynthetic relative of the apicomplexan parasites that shares a common ancestor with Symbiodinium. To explore the nature of the association between C. velia and corals we first isolated C. velia from the coral Montipora digitata and then exposed aposymbiotic Acropora digitifera and A. tenuis larvae to these cultures. Three C. velia cultures were isolated, and symbiosis was established in coral larvae of both these species exposed to all three clones. Histology verified that C. velia was located in the larval endoderm and ectoderm. These results indicate that C. velia has the potential to be endosymbiotic with coral larvae., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The TGF-b superfamily cytokine MIC-1/GDF15 circulates in all humans and when overproduced in cancer leads to anorexia/cachexia, by direct action on brain feeding centres. In these studies we have examined the role of physiologically relevant levels of MIC-1/GDF15 in the regulation of appetite, body weight and basal metabolic rate. MIC-1/GDF15 gene knockout mice (MIC-1(-/-)) weighed more and had increased adiposity, which was associated with increased spontaneous food intake. Female MIC-1(-/-) mice exhibited some additional alterations in reduced basal energy expenditure and physical activity, possibly owing to the associated decrease in total lean mass. Further, infusion of human recombinant MIC-1/GDF15 sufficient to raise serum levels in MIC-1(-/-) mice to within the normal human range reduced body weight and food intake. Taken together, our findings suggest that MIC-1/GDF15 is involved in the physiological regulation of appetite and energy storage.

We report the case of a patient highly susceptible to invasive aspergillosis who received treatment with voriconazole (VRC). As part of therapeutic drug monitoring, VRC plasma trough concentrations were measured, showing undetectable levels (<0.16 µg/ml). Genotyping showed a heterozygous profile CYP2C19*1/*17, known to be associated with an ultrarapid-metabolism phenotype, contributing to the very low systemic exposure observed. Therefore, in this situation, the use of VRC treatment could be associated with therapeutic failure.

This study aimed to purify and characterize the peanut allergens Ara h1 and Ara h3 from four cultivars that represent the four major market types to provide better understanding of the molecular organization of oligomers in different market types. The chromatographic profiles of Ara h1 and Ara h3 from the four cultivars obtained from anion exchange chromatography were similar. However, they differed in the distribution of trimeric and hexameric structures of Ara h3 isolated by size exclusion chromatography. The Menzies (Runner market type) and Walter (Spanish market type) cultivars, wherein Ara h3 proteins consist of two acidic subunits, exhibited trimeric and hexameric conformations proportionally. However, the Middleton (Virginia market type) and Kelinci (Valencia market type) cultivars, wherein Ara h3 proteins consist of three acidic subunits, showed predominantly a hexameric structure. The oligomeric structures of the purified Ara h1 demonstrated strong IgE binding properties, whereas the allergenic property of the oligomeric Ara h3 could not be performed due to lack of availability of specific IgE. In addition, the polyclonal antibodies raised against the purified Ara h1 and Ara h3 showed highly specific binding to their respective antigens.

Background: The stump impingement reflex is a subtle bounce to the knee thought to be caused by hamstring contraction when the knee is brought into extension and the torn ACL stump impinges between the distal femur and the tibial plateau. We have studied the diagnostic value of this sign., Findings: From Feb 2008-Feb 2009, we audited 30 patients who underwent urgent arthroscopy for acutely locked knees. The presence of the stump impingement reflex prior to surgery was compared with the intra-operative findings. The diagnostic values of the stump impingement sign were found to be: Sensitivity 58%, Specificity 81%, Positive predictive value 70%, Negative predictive value 72% and Accuracy 71%., Conclusions: We believe that the stump impingement reflex is a specific sign for ACL stump impingement as a cause of knee locking. We recommend close inspection for this sign when examining locked knees.

Exposure to fetal bovine serum (FBS) is shown herein to reduce the aggregate size of titanium dioxide (TiO(2)) nanoparticles, affecting uptake and consequent effect on A549 and H1299 human lung cell lines. Initially, the cellular uptake of the FBS-treated TiO(2) was lower than that of non-FBS-treated TiO(2). Expulsion of particles was then observed, followed by a second phase of uptake of FBS-treated TiO(2), resulting in an increase in the cellular content of FBS-treated TiO(2), eventually exceeding the amount by cells exposed to non-FBS-treated TiO(2). Surface adsorbed vitronectin and the clathrin-mediated endocytosis pathway were shown to regulate the uptake of TiO(2) into A549 cells, while the endocytosis mechanism responsible remains elusive for H1299. Intriguingly, nystatin treatment was shown to have the unexpected effect of increasing nanoparticle uptake into the A549 cells via an alternate endocytic pathway. The surface adsorbed serum components were found to provide some protection from the cytotoxic effect of endocytosed TiO(2) nanoparticles.

Background: Elevated macrophage inhibitory cytokine-1 (MIC-1/GDF15) levels in serum mediate anorexia and weight loss in some cancer patients and similarly elevated levels occur in chronic kidney disease (CKD). Serum MIC-1/GDF15 is also elevated in chronic inflammatory diseases and predicts atherosclerotic events independently of traditional risk factors. The relationship between chronic inflammation, decreasing body mass index (BMI) and increased mortality in CKD is not well understood and is being actively investigated. MIC-1/GDF15 may link these features of CKD., Methods: Cohorts of incident dialysis patients from Sweden (n = 98) and prevalent hemodialysis patients from the USA (n = 381) had serum MIC-1/GDF15, C-reactive protein (CRP) levels and BMI measured at study entry. Additional surrogate markers of nutritional adequacy, body composition and inflammation were assessed in Swedish patients. Patients were followed for all-cause mortality., Results: In the Swedish cohort, serum MIC-1/GDF15 was associated with decreasing BMI, measures of nutrition and markers of oxidative stress and inflammation. Additionally, high serum MIC-1/GDF15 levels identified patients with evidence of protein-energy wasting who died in the first 3 years of dialysis. The ability of serum MIC-1/GDF15 to predict mortality in the first 3 years of dialysis was confirmed in the USA cohort. In both cohorts, serum MIC-1/GDF15 level was an independent marker of mortality when adjusted for age, CRP, BMI, history of diabetes mellitus and/or cardiovascular disease and glomerular filtration rate or length of time on dialysis at study entry., Conclusions: MIC-1/GDF15 is a novel independent serum marker of mortality in CKD capable of significantly improving the mortality prediction of other established markers. MIC-1/GDF15 may mediate protein-energy wasting in CKD and represent a novel therapeutic target for this fatal complication.

A number of studies have shown that, as a result of the ambiguity of US legal mandates, organizations have considerable latitude in how they comply with regulations. In this paper, we address how the different agendas of the federal and state governments increase ambiguities in state-firm relations and how states are interested actors in creating opportunities for firms to navigate the federal legislation. We analyze the institutional forces behind bank acquisitions within and across state lines in order to illuminate the ways that US states take advantage of federal ambiguity and are able to shape corporate practices to their benefit. We specifically examine how patterns of bank acquisitions are shaped by the crucial relationship between the federal Community Reinvestment Act (CRA) and a little-understood provision in the federal tax code that is implemented at the state level, the Low-Income Housing Tax Credit (LIHTC). The relationship is complex because, while the federal government uses the CRA to control bank acquisition activity, states promote use of the LIHTC, through which banks can address federal CRA concerns, and thereby promote bank acquisitions in their jurisdictions. Thus, our findings suggest that the implementation of social legislation at one level in a federal regulatory system undermines the mechanisms of social legislation at another level. We use archival research and in-depth interviews to examine the interaction between these institutional processes and formulate hypotheses that predict the ways in which bank acquisitions are constrained by banks' CRA ratings and the way states in turn help banks overcome their CRA constraints. Quantitative analyses of all bank acquisitions in the United States from 1990-2000 largely support these hypotheses., (Copyright © 2011 Elsevier Inc. All rights reserved.)