Revealing the Toxicity of Chlorate Through the Analysis of Molecular Interaction using Viscosity Measurements and Apparent Molar Volumes

While chlorate has the ability to induce flowering in longan, it also has adverse impacts on the crop. Revealing the toxicity of chlorate in the environment is more than just about the environment and about human health, as well.Because of the large introduction of this chemical into the environment from the paper processing industry, there is indeed a lot of concern about its toxicity. Chlorate toxicology in the longan plant has been thoroughly investigated in solutions using viscosities and apparent molar volumes. The hydration of molecules and volume changes are involved in various chemical and biological processes in plant tissues, and their complete understanding demands a good idea for volumetric and viscometric study. It offers good data acquisition techniques for solute, solvent and solvent-solvent interactions. Multi-component systems containing KClO3+ water + ionic solid (ionic solids = KCl, KNO3 ,NH4NO3 and KH2PO4, are currently being worked out to study the dependence of transport properties of potassium chlorate in aqueous electrolyte solutions, with concentrations and temperature of solutions. The assessed kd values are used to predict whether the solvolysis of KClO3 in the presence of other electrolytes is a quick or slow process.

Enoxaparin and Pentosan Polysulfate Bind to the SARS-CoV-2 Spike Protein and Human ACE2 Receptor, Inhibiting Vero Cell Infection

As with many other pathogens, SARS-CoV-2 cell infection is strongly dependent on the interaction of the virus-surface Spike protein with the glycosaminoglycans of target cells. The SARS-CoV-2 Spike glycoprotein was previously shown to interact with cell-surface-exposed heparan sulfate and heparin in vitro. With the aim of using Enoxaparin as a treatment for COVID-19 patients and as prophylaxis to prevent interpersonal viral transmission, we investigated GAG binding to the Spike full-length protein, as well as to its receptor binding domain (RBD) in solution by isothermal fluorescence titration. We found that Enoxaparin bound to both protein variants with similar affinities, compared to the natural GAG ligand heparan sulfate (with Kd-values in the range of 600–680 nM). Using size-defined Enoxaparin fragments, we discovered the optimum binding for dp6 or dp8 for the full-length Spike protein, whereas the RBD did not exhibit a significant chain-length-dependent affinity for heparin oligosaccharides. The soluble ACE2 receptor was found to interact with unfractionated GAGs in the low µM Kd range, but with size-defined heparins with clearly sub-µM Kd-values. Interestingly, the structural heparin analogue, pentosan polysulfate (PPS), exhibited high binding affinities to both Spike variants as well as to the ACE2 receptor. In viral infection experiments, Enoxaparin and PPS both showed a strong inhibition of infection in a concentration range of 50–500 µg/mL. Both compounds were found to retain their inhibitory effects at 500 µg/mL in a natural biomatrix-like human sputum. Our data suggest the early topical treatment of SARS-CoV-2 infections with inhaled Enoxaparin; some clinical studies in this direction are already ongoing, and they further imply an oral or nasal prophylactic inactivation of the virus by Enoxaparin or PPS for the prevention of inter-personal viral transmission.

Assessment of Sediment Arsenic and Iron Occurrence and Leaching Potential in a Potable Water Treatment Wastewater Stabilization Pond System

Wastewater stabilization ponds (WSPs) are commonly used to reduce wastewater metal(loid) concentrations from drinking water treatment plants (DWTPs) through sedimentation. However, this results in increased sediment concentrations that can be released back into the overlying water. Thus, our goal was to evaluate the WSP metal(loid)s occurrence and leaching potential. Currently, a Saskatchewan based DWTP’s WSP system was investigated given historically elevated effluent As and Fe concentrations. The WSP consists of five ponds that were sampled on six occasions in 2019 and 2020. In addition, sediments were used in laboratory-based experiments to determine their leaching potential. Overall, the sediments were found to contain elevated concentrations of As and Fe with 25 to 400 and 10,000 to 45,000 mg/kg, respectively. Leaching experiments indicated that the pond sediments could potentially release As and Fe with log Kd values ranging from 2.21 to 4.31 L/kg, while Fe ranged from 3.32 to 5.53 L/kg.

Rosmarinic Acid and Sodium Citrate Have a Synergistic Bacteriostatic Effect against Vibrio Species by Inhibiting Iron Uptake

Background: New strategies are needed to combat multidrug-resistant bacteria. The restriction of iron uptake by bacteria is a promising way to inhibit their growth. We aimed to suppress the growth of Vibrio bacterial species by inhibiting their ferric ion-binding protein (FbpA) using food components. Methods: Twenty spices were selected for the screening of FbpA inhibitors. The candidate was applied to antibacterial tests, and the mechanism was further studied. Results: An active compound, rosmarinic acid (RA), was screened out. RA binds competitively and more tightly than Fe3+ to VmFbpA, the FbpA from V. metschnikovii, with apparent KD values of 8 μM vs. 17 μM. Moreover, RA can inhibit the growth of V. metschnikovii to one-third of the control at 1000 μM. Interestingly, sodium citrate (SC) enhances the growth inhibition effect of RA, although SC only does not inhibit the growth. The combination of RA/SC completely inhibits the growth of not only V. metschnikovii at 100/100 μM but also the vibriosis-causative pathogens V. vulnificus and V. parahaemolyticus, at 100/100 and 1000/100 μM, respectively. However, RA/SC does not affect the growth of Escherichia coli. Conclusions: RA/SC is a potential bacteriostatic agent against Vibrio species while causing little damage to indigenous gastrointestinal bacteria.

Selection and characterisation of single-stranded DNA aptamers for triclosan

<p>Triclosan (TCS) is a chlorinated organic compound which, due to its antibacterial properties in vitro, has found widespread usages in many medical and consumer products such as textiles, plastics and personal care products. Humans are directly and chronically exposed to TCS via dermal and mucosal contact from the use of TCS-formulated products such as soap and toothpaste. TCS is classified as an environmental contaminant by the European Union Water Framework Directive, whose mandatory goal is to develop new and simple-to-use analytical methodologies capable of measuring low concentrations of TCS and that are suitable for high-throughput detection. Synthetically-derived single-stranded oligonucleotides, also known as aptamers, are superior candidates for the development of sensitive and high-throughput biosensing strategies. Biosensors utilising aptamers as molecular recognition elements have showed great promise in a variety of diagnostic and therapeutic applications, especially for the detection of small molecular weight organic compounds such as TCS. The aim of this thesis was to develop aptamers as new capture reagents for TCS, as the first step towards the development of an alternative, user-friendly, diagnostic technique for monitoring TCS in both environmental and biological samples. The objectives of the thesis were to: [i] produce by in vitro selection procedures, TCS binding single-stranded DNA (ssDNA) aptamers; [ii] characterise the selected aptamers and determine their equilibrium dissociation constant (Kd) values and; [iii] evaluate the applicability of the selected aptamers in a biosensing platform. To achieve these objectives, ssDNA aptamers capable of binding TCS were generated in vitro using a sequential approach known as systematic evolution of ligands by exponential enrichment (SELEX). An affinity column-based SELEX strategy together with a variety of SELEX modifications such as negative and counter selections, real-time amplification and fluorescence quantification were explored for finding TCS specific aptamers. A total of 20 TCS aptamers, ten from 8 rounds of a basic-SELEX procedure, and the other ten from 10 rounds of a revised-SELEX procedure were generated. In general, these aptamers showed acceptable levels of sensitivity and specificity to TCS, and the best binding aptamer demonstrated a Kd value of 378 nM. The Kd value is comparable to published Kd values for compounds that share similar chemical structures to TCS. In addition, a novel fluorescent-based imaging method was developed in this dissertation. The method developed provides an alternative approach for monitoring SELEX progression and has the potential to simplify the way to characterise the binding properties of an aptamer to its cognate target. The utility of this method was compared with commonly used methods such as dot blot and fluorescent binding assays. The performance of the new imaging method was superior to the existing methods in terms of accuracy, simplicity and reproducibility. Furthermore, the best binding TCS aptamer was evaluated for its utility in an aptamer-based biosensor. The developed aptasensor, utilising a TCS aptamer as the recognition element and gold nanoparticles (AuNPs) as the signal reporter, was capable of detecting TCS in spiked-water samples at concentrations ranging from 20–750 nM with a visual detection limit of 150 nM. In conclusion, methods were developed to select, refine, and characterise ssDNA aptamers capable of binding to TCS, and these aptamers have the potential to offer a sensitive, simple-to-use, and user-friendly analytical method for TCS detection.</p>

Selection and characterisation of single-stranded DNA aptamers for triclosan

<p>Triclosan (TCS) is a chlorinated organic compound which, due to its antibacterial properties in vitro, has found widespread usages in many medical and consumer products such as textiles, plastics and personal care products. Humans are directly and chronically exposed to TCS via dermal and mucosal contact from the use of TCS-formulated products such as soap and toothpaste. TCS is classified as an environmental contaminant by the European Union Water Framework Directive, whose mandatory goal is to develop new and simple-to-use analytical methodologies capable of measuring low concentrations of TCS and that are suitable for high-throughput detection. Synthetically-derived single-stranded oligonucleotides, also known as aptamers, are superior candidates for the development of sensitive and high-throughput biosensing strategies. Biosensors utilising aptamers as molecular recognition elements have showed great promise in a variety of diagnostic and therapeutic applications, especially for the detection of small molecular weight organic compounds such as TCS. The aim of this thesis was to develop aptamers as new capture reagents for TCS, as the first step towards the development of an alternative, user-friendly, diagnostic technique for monitoring TCS in both environmental and biological samples. The objectives of the thesis were to: [i] produce by in vitro selection procedures, TCS binding single-stranded DNA (ssDNA) aptamers; [ii] characterise the selected aptamers and determine their equilibrium dissociation constant (Kd) values and; [iii] evaluate the applicability of the selected aptamers in a biosensing platform. To achieve these objectives, ssDNA aptamers capable of binding TCS were generated in vitro using a sequential approach known as systematic evolution of ligands by exponential enrichment (SELEX). An affinity column-based SELEX strategy together with a variety of SELEX modifications such as negative and counter selections, real-time amplification and fluorescence quantification were explored for finding TCS specific aptamers. A total of 20 TCS aptamers, ten from 8 rounds of a basic-SELEX procedure, and the other ten from 10 rounds of a revised-SELEX procedure were generated. In general, these aptamers showed acceptable levels of sensitivity and specificity to TCS, and the best binding aptamer demonstrated a Kd value of 378 nM. The Kd value is comparable to published Kd values for compounds that share similar chemical structures to TCS. In addition, a novel fluorescent-based imaging method was developed in this dissertation. The method developed provides an alternative approach for monitoring SELEX progression and has the potential to simplify the way to characterise the binding properties of an aptamer to its cognate target. The utility of this method was compared with commonly used methods such as dot blot and fluorescent binding assays. The performance of the new imaging method was superior to the existing methods in terms of accuracy, simplicity and reproducibility. Furthermore, the best binding TCS aptamer was evaluated for its utility in an aptamer-based biosensor. The developed aptasensor, utilising a TCS aptamer as the recognition element and gold nanoparticles (AuNPs) as the signal reporter, was capable of detecting TCS in spiked-water samples at concentrations ranging from 20–750 nM with a visual detection limit of 150 nM. In conclusion, methods were developed to select, refine, and characterise ssDNA aptamers capable of binding to TCS, and these aptamers have the potential to offer a sensitive, simple-to-use, and user-friendly analytical method for TCS detection.</p>

Deciphering the TLT-1 Fibrinogen Ligand Interaction

Background: Platelets, derived from megakaryocytes primarily play a central role in thrombosis and hemostasis, however, they extend beyond this role as immune cells that initiate and accelerate various vascular inflammatory conditions. Upon activation, platelets release TREM-Like Transcript-1 (TLT-1) from their a-granules onto their surface. Early studies by amino link columns preloaded with soluble TLT-1 followed by mass spectrometry and immunoblotting identified fibrinogen as a ligand for TLT-1. Fibrinogen is a plasma protein that is essential for clot formation, during inflammation and hypercoagulable states tissue deposition and plasma concentration of fibrinogen are increased, demonstrating a role of fibrinogen in both thrombosis and inflammation. TLT-1 binding fibrinogen was a surprising discovery since αIIbβ3, the most abundant platelet receptor, also binds fibrinogen and facilitates platelet aggregation. It is difficult to understand why there are two platelet specific receptors that have the same ligand, drawing us to question what the difference in function between the two is? Our studies suggest that although TLT-1 may assist in clot formation and hemostasis to arrest bleeding in a non-inflammatory setting like αIIbβ3, TLT-1's main association is with regulating inflammatory-derived bleeding. Very little is known about the TLT-1-Fibrinogen interaction, further studies would set the stage for a better understanding as to why two fibrinogen ligands exist on platelets and potentially outline a novel platelet therapeutic target during hypercoagulable and/or hyperinflammatory states. We set out to determine the binding affinity and localize the binding sites for the TLT-1 fibrinogen molecular interaction. Aims: Delineate the TLT-1 fibrinogen molecular interaction and elucidate the mechanism by which this interaction drives inflammation and thrombosis-hemostasis. Methods: To confirm the TLT-1 fibrinogen ligand interaction we carried out a kinetics assay using an Octet Qk e Bio-layer Interferometry (BLI) that measures biomolecular complex formation in real time. The TLT-1 Chimera was captured onto an Anti-Human Fc Capture (AHC) Biosensor, washed in kinetics buffer to limit nonspecific binding and submerged in a 96 well plate containing varying concentrations of Fibrinogen. To localize the exact binding sites for this molecular interaction, we digested fibrinogen using trypsin and carried out an immunoprecipitation (IP) followed by Liquid Chromatography-Mass Spectrometry (LC-MS/MS). Results: The curve (Figure 1) shows that the TLT-1 fibrinogen interaction has increasing bimolecular complex formation with increases in concentration of Fibrinogen (15.625nM - 250nM), with a concentration of 250nM showing the best bicomplex formation. In the control well with HIV01 4E10 capture, reference and sensor well, no bicomplex formation is shown, highlighting the specificity of the TLT-1 fibrinogen interaction. The curve illustrates a strong association with no dissociation, suggesting a strong interaction between the proteins. We isolated and identified four potential peptides (Alpha chain: GGSTSYGTGSETESPR, GSESGIFTNTK, Beta chain: QDGSVDFGR , QGFGNVATNTDGK) that bind TLT-1. We are currently performing BLI Competitive kinetics assays using biotinylated constructs of the peptides isolated from the Immunoprecipitation/ LC-MS/MS. The BLI competitive assays using the four peptides are suggestive of an interaction between TLT-1 and the four peptides as illustrated by increasing bimolecular complex formation with increasing concentration of soluble TLT-1 for all four peptides(data not shown). Conclusions: We obtained an equilibrium dissociation constant (KD) of 3.02 ± 0.20 nM for the TLT-1 fibrinogen interaction, suggesting a high affinity interaction between TLT-1 and fibrinogen. In our preliminary results from the BLI Competitive kinetics assays we obtained KD values within the nanomolar concentration range and are currently conducting experiments to optimize conditions to obtain our final bicomplex binding curve and KD values. We are currently assessing the identified peptides for potential of mediating the molecular interaction between TLT-1 and fibrinogen. Our poster will report the current state of these studies . Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Diffuse Attenuation Coefficient (Kd) from ICESat-2 ATLAS Spaceborne Lidar Using Random-Forest Regression

This study investigates a new method for measuring water turbidity—specifically, the diffuse attenuation coefficient of downwelling irradiance Kd —using data from a spaceborne, green-wavelength lidar aboard the National Aeronautics and Space Administration's ICESat-2 satellite. The method enables us to fill nearshore data voids in existing Kd data sets and provides a more direct measurement approach than methods based on passive multispectral satellite imagery. Furthermore, in contrast to other lidar-based methods, it does not rely on extensive signal processing or the availability of the system impulse response function, and it is designed to be applied globally rather than at a specific geographic location. The model was tested using Kd measurements from the National Oceanic and Atmospheric Administration's Visible Infrared Imaging Radiometer Suite sensor at 94 coastal sites spanning the globe, with Kd values ranging from 0.05 to 3.6 m –1 . The results demonstrate the efficacy of the approach and serve as a benchmark for future machine-learning regression studies of turbidity using ICESat-2.

Tuning the sensitivity of genetically encoded fluorescent potassium indicators through structure-guided and genome mining strategies

Genetically encoded potassium indicators lack optimal binding affinity for monitoring intracellular dynamics in mammalian cells. Through structure-guided design and genome mining of potassium binding proteins, we developed green fluorescent potassium indicators with a broad range of binding affinities. KRaION1, based on the insertion of a potassium binding protein (Ec-Kbp) into the fluorescent protein mNeonGreen, exhibits an isotonically measured Kd of 69±10 (mM; mean ± standard deviation used throughout). We identified Ec-Kbp's binding site using NMR spectroscopy to detect protein-thallium scalar couplings and refined the structure of Ec-Kbp in its potassium-bound state. Guided by this structure, we modified KRaION1, yielding KRaION2, which exhibits an isotonically measured Kd of 96±9 (mM). We identified four Ec-Kbp homologs as potassium binding proteins, which yielded indicators with isotonically measured binding affinities in the 39-112 (mM) range. KRaIONs expressed and functioned in HeLa cells, but exhibited lower Kd values, which were mirrored by lower Kd values measured in vitro when holding sodium constant. Thus, potassium indicator Kd may need to be evaluated in the context of a given experimental goal.

Pharmaceuticals and Their Main Metabolites in Treated Sewage Sludge and Sludge-Amended Soil: Availability and Sorption Behaviour

This work evaluated the availability and sorption behaviour of four pharmaceuticals and eight of their metabolites in sewage sludge and sludge-amended soil. Digested sludge and compost were evaluated. The highest levels found in digested sludge corresponded to caffeine (up to 115 ng g−1 dm), ibuprofen (45 ng g−1 dm) and carbamazepine (9.3 ng g−1 dm). The concentrations measured in compost were even lower than in digested sludge. No compound was detected in sludge-amended soils. This fact could be due to the dilution effect after sludge application to soil. Different adsorption capacities in sludge–soil mixtures were measured for the studied compounds at the same spike concentration. In general, except for paraxanthine and 3-hydroxycarbamazepine, the metabolite concentrations measured in the mixtures were almost two-fold lower than those of their parent compounds, which can be explained by their mobility and lixiviation tendency. The log Kd ranged from −1.55 to 1.71 in sludge samples and from −0.29 to 1.18 in soil–sludge mixtures. The log Kd values calculated for compost were higher than those calculated for digested sludge. The obtained results implied that the higher organic carbon content of compost could influence soil contamination when it is applied to soil.