Site-specific Activity Research Articles

Establishment of a human embryonic stem cell line, WAe009-A-99, with constitutive expression of the dCas9-p300 fusion protein

CRISPR/Cas9-based transcriptional regulation systems can induce the site-specific activation or repression of endogenous genes. p300 is a transcriptional co-activator that functions as a histone acetyltransferase that regulates gene transcription via chromatin remodeling. Here, we generated a human embryonic stem cell line stably expressing catalytically dead Cas9 (dCas9) fused to the catalytic core domain of human p300 via lentiviral transduction. This cell line can be used for locus-specific histone acetylation in combination with guide RNAs, and is a valuable tool for gene regulation in stem cell research.

CD25 As Feedback Regulator of Oncogenic TCR Signaling in T-Cell Lymphoma

Introduction: CD25, also known as interleukin-2 receptor α chain (IL-2Rα), forms a heterotrimer with IL-2Rβ and common γ chain, and mediates IL-2 signaling. IL-2 signaling is essential for T- and NK-cell survival and proliferation. Additionally, CD25 is required for development of thymic regulatory T cells (Treg) and their suppressive function. Interestingly, CD25 is upregulated in a variety of lymphoid and myeloid malignancies, which do not depend on IL-2 signaling, suggesting IL-2-independent roles of CD25. Here, we seek to investigate whether CD25 has previously unrecognized functions in T cells and T-cell lymphomas. Results: Our previous study in B cells has discovered the cytoplasmic tail of CD25 as an essential structural element for site-specific recruitment of inhibitory phosphatases. CD25-mediated shuttling of inhibitory phosphatases balanced strength of oncogenic B-cell receptor (BCR)-signaling and was essential for cell-survival in B-cell malignancies. Interestingly, we found that CD25 expressed on B cells was monomeric and showed dynamic recruitment to BCR or its oncogenic mimics within lipid rafts. Studying gene expression data across different hematopoietic lineages, we found CD25 was strongly induced in T cell receptor (TCR)-activated T cells, as well as in T-cell malignancies that harbor oncogenic TCR-mimics, such as PTCL. Our mechanistic studies discovered that the serine residue 268 (S268) in the cytoplasmic tail of CD25 was a principal substrate of protein kinase C (PKC), and that S268 phosphorylation by PKC was critical for cell surface expression of CD25. The proximity-based labeling assays (Bio-ID) revealed that CD25-S268 but not its phospho-dead mutant recruits PKC and its adapter RACK1, which scaffold the inhibitory phosphatases SHIP1 and SHP1 for site-specific activation in lipid rafts. CRISPR-mediated knock-in of CD25-S268A that abrogates the formation of inhibitory phosphatase complexes in primary human T cells enabled us to specifically interrogate the functional relevance of CD25-S268 in the regulation of TCR signaling. By using single stranded homology-directed repair templates (ssHDRT) that contain a Cas9 shuttling sequence, we were able to achieve 70% knock-in efficiency in primary human T cells (Figure 1). Signaling studies with these cells revealed that S268A mutation significantly augmented TCR signaling strength. Additionally, S268A mutation resulted in acute inactivation of inhibitory phosphatases including SHP1. Consistent with these results, CD25 shuttling to TCR complex was promptly induced upon TCR stimulation and was mitigated by S268 mutation. Furthermore, immunophenotyping showed that loss of S268 phosphorylation resulted in increased expression of T-cell activation markers such as CD44 and CD69. Based on the ex vivo results, we generated a transgenic mouse model with germline knock-in of CD25-S268A, which would allow us to study S268-specific roles in CD25-mediated feedback regulation of TCR and oncogenic signaling in vivo. Conclusions: The role of CD25 in mediating IL-2 signaling has been extensively studied. However, increasing evidence suggests that CD25 plays a more sophisticated role in TCR signaling and T-lymphomagenesis. Here we discovered a previously unrecognized function of CD25 as a feedback regulator of TCR signaling. TCR activation or oncogenic TCR-mimics induce CD25-mediated recruitment of inhibitory phosphatases in lipid rafts to mitigate TCR signaling or curb excessive oncogenic signaling. Figure 1. CRISPR-mediated knock-out of CD25 alone, or followed by knock-in of CD25-S268-GFP or CD25-A268-GFP, in primary human T cells. The proportion of CD25-expressing GFP+ cells are analyzed by flow cytometry on day 4 and day 30 post CRISPR. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Complementation Dependent Enzyme Prodrug Therapy Enables Targeted Activation of Prodrug on HER2-Positive Cancer Cells.

Antibodies have been explored for decades for the delivery of small molecule cytotoxins directly to diseased cells. In antibody-directed enzyme prodrug therapy (ADEPT), antibodies are armed with enzymes that activate nontoxic prodrugs at tumor sites. However, this strategy failed clinically due to off-target toxicity associated with the enzyme prematurely activating prodrug systemically. We describe here the design of an antibody-fragment split enzyme platform that regains activity after binding to HER2, allowing for site-specific activation of a small molecule prodrug. We evaluated a library of fusion constructs for efficient targeting and complementation to identify the most promising split enzyme pair. The optimal pair was screened for substrate specificity among chromogenic, fluorogenic, and prodrug substrates. Evaluation of this system on HER2-positive cells revealed 7-fold higher toxicity of the activated prodrug over prodrug treatment alone. Demonstrating the potential of this strategy against a known clinical target provides the basis for a unique therapeutic platform in oncology.

Determining baseline radiation levels in marine biota – A comparison of SE Queensland commercial species

Limited data exists on the contribution to radiological dose to members of the public from ingestion of radioactivity in seafood in the Australian diet. There is also a lack of data available to assess the radiological dose to marine fauna in Australian waters. Natural and anthropogenic radionuclides and trace metals were measured in the edible flesh of prawns to determine the radiological dose to humans. The remaining tissues were combined and analysed to enable uptake and environmental radiological doses to be assessed. Although in international studies, the edible flesh is generally measured to determine radiation dose or ingestion of trace metals, the effects of preparation and cooking techniques are rarely assessed. In this study, cooking and preparation techniques that may influence the radiological dose to humans were assessed. Eggs were also removed from a selected number of samples to assess the potential dose in sensitive developmental tissues and possible implications for environmental effects. Order of magnitude differences in 210Po activity concentrations and Cd concentrations were observed between whole animals and the edible flesh of Australian caught King (Melicertus spp.) and Tiger (Penaeus spp.) prawns, with the hepatopancreas primarily responsible for this difference. Most 210Po was unsupported by 210Pb and activity concentrations of all other radionuclides measured (137Cs, 210Pb, 226Ra, 232Th, and 238U) were very low. The major contribution to radiation dose via the ingestion pathway and to the organism itself was from 210Po. Cooking techniques that may lead to leaching of 210Po from the hepatopancreas could substantially increase the radiological dose from ingestion of this isotope. Organism dose estimates using different input assumptions in the radiological assessment tool “ERICA”, including site-specific tissue activity concentrations with site- or region-specific media concentrations, were compared with ERICA default distribution coefficients (Kd) and concentration ratios.

Hibiscus sabdariffa Extract Protects HaCaT Cells against Phenanthrene-Induced Toxicity through the Regulation of Constitutive Androstane Receptor/Pregnane X Receptor Pathway.

Phenanthrene (Phe) exposure is associated with skin ageing, cardiotoxicity and developmental defects. Here, we investigated the mode of Phe toxicity in human keratinocytes (HaCaT cells) and the attenuation of toxicity on pre-treatment (6 h) with ethanol extract of Hibiscus sabdariffa calyxes (HS). Cell viability, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨm) alteration, changes in the transcriptional activity of selected genes involved in phase I and II metabolism, antioxidant response and gluconeogenesis, western blot and docking studies were performed to determine the protective effect of HS against Phe. Phe (250 μM) induced cytotoxicity in HaCaT cells through AhR-independent, CAR/PXR/RXR-mediated activation of CYP1A1 and the subsequent alterations in phase I and II metabolism genes. Further, CYP1A1 activation by Phe induced ROS generation, reduced ΔΨm and modulated antioxidant response, phase II metabolism and gluconeogenesis-related gene expression. However, pre-treatment with HS extract restored the pathological changes observed upon Phe exposure through CYP1A1 inhibition. Docking studies showed the site-specific activation of PXR and CAR by Phe and inhibition of CYP1A1 and CYP3A4 by the bioactive compounds of HS similar to that of the positive controls tested. Our results conclude that HS extract can attenuate Phe-induced toxicity in HaCaT cells through CAR/PXR/RXR mediated inhibition of CYP1A1.

Catecholaminergic cell type-specific expression of Cre recombinase in knock-in transgenic rats generated by the Combi-CRISPR technology

BackgroundCell groups containing catecholamines provide a useful model to study the molecular and cellular mechanisms underlying the morphogenesis, physiology, and pathology of the central nervous system. For this purpose, it is necessary to establish a system to induce catecholaminergic group-specific expression of Cre recombinase. Recently, we introduced a gene cassette encoding 2A peptide fused to Cre recombinase into the site between the C-terminus and translational termination codons of the rat tyrosine hydroxylase (TH) open reading frame by the Combi-CRISPR technology, which is a genomic editing method to enable an efficient knock-in (KI) of long DNA sequence into a target site. However, the expression patterns of the transgene and its function as well as the effect of the mutation on the biochemical and behavioral phenotypes in the KI strains have not been characterized yet. New methodWe aimed to evaluate the usefulness of TH-Cre KI rats as an experimental model for investigating the structure and function of catecholaminergic neurons in the brain. ResultsWe detected cell type-specific expression of Cre recombinase and site-specific recombination activity in the representative catecholaminergic groups in the TH-Cre KI rat strains. In addition, we measured TH protein levels and catecholamine accumulation in the brain regions, as well as motor, reward-related, and anxiety-like behaviors, indicating that catecholamine metabolism and general behavior are apparently normal in these KI rats. ConclusionsTH-Cre KI rat strains produced by the Combi-CRISPR system offer a beneficial model to study the molecular and cellular mechanics for the morphogenesis, physiology, and pathology of catecholamine-containing neurons in the brain.

Targeting application of hierarchical porous carbon by facile one-step site-specific activation of wood powder: CO2 adsorption and supercapacitors

Optimization of pore parameters for specific applications is crucial for utilization of hierarchical porous carbon (HPC). In this work, we report a facile method to obtain HPCs for targeting applications in a controlled manner by a one-step site-specific activation method with trace amounts of KOH. For CO2 adsorption applications, the material is optimized to have a pore size consistent with the kinetic diameter of CO2, which facilitates the diffusion of CO2 in the material. The optimized material shows exceptional CO2 adsorption capacity (5.21 mmol g−1 at 0 °C and 3.54 mmol g−1 at 25 °C), and excellent adsorption reversibility (97.6% at 0 °C and 98.9% at 25 °C). For supercapacitor applications, the pore size of the optimized HPC is multiple of the size of hydrated potassium ions, which enhances the transport of electrolyte ions. The optimized material provides a high specific capacitance of 369 F g−1 at 0.5 A g−1, and the assembled symmetric supercapacitor exhibits an extraordinary energy density of 30.51 Wh kg−1 at a power density of 92.23 W kg−1. This work provides new insight into tuning of the pore parameters for specific applications of HPCs, and a facile strategy for value-added utilization of biomass materials.

Hydroxamic Acid-Modified Peptide Library Provides Insights into the Molecular Basis for the Substrate Selectivity of HDAC Corepressor Complexes.

Targeting the lysine deacetylase activity of class I histone deacetylases (HDACs) is potentially beneficial for the treatment of several diseases including human immunodeficiency virus (HIV) infection, Alzheimer’s disease, and various cancers. It is therefore important to understand the function and mechanism of action of these enzymes. Class I HDACs act as catalytic components of seven large, multiprotein corepressor complexes. Different HDAC corepressor complexes have specific, nonredundant roles in the cell. It is likely that their specific functions are at least partly influenced by the substrate specificity of the complexes. To address this, we developed chemical tools to probe the specificity of HDAC complexes. We assessed a library of acetyl-lysine-containing substrate peptides and hydroxamic acid-containing inhibitor peptides against the full range of class I HDAC corepressor complexes. The results suggest that site-specific HDAC corepressor complex activity is driven in part by the recognition of the primary amino acid sequence surrounding a particular lysine position in the histone tail.

Formulation and Evaluation of Eudragit L- 100 based Nanoparticles of Senna for Treatment of Constipation

Background: The pharmaceutical industries are keen in development of new herbal bioactive molecules with an improved effect and less toxicity in order to replace synthetic drugs. Constipation is the most prevalent medical concerns. It’s estimated that one in five adults worldwide suffers from constipation. Hence, the demand of herbal laxatives is increasing worldwide. The purpose of this research work was to develop Senna nanoparticles with the help of Eudragit L100 an enteric coating polymer to provide site specific activity and to prevent degradation of Senna in acidic environment. Materials and Methods: Senna loaded nanoparticles were prepared by using emulsion solvent evaporation method. Different formulation has been formulated with varying concentration of Eudragit L-100 and constant aqueous and organic phase ratio (1:9). Drug and polymer compatibility study was analysed by FTIR. The prepared formulation was characterized by melting point, particle size, zeta potential, polydispersity index, vesical morphology and in-vitro drug release. Results: All the formulation batches shows particle size between 380.61 - 1061.71nm. The zeta of optimized batch (F5) was found to be -23.75mV. Entrapment efficiency of the entire prepared formulation batch in between 3.16±0.31- 55.18±0.35%. The in-vitro drug release shows maximum drug release i.e. 61.03% in 8 hrs which shows that release of drug prolonged and site specific due to use of enteric coated polymer Eudragit L100. Conclusion: The developed Senna loaded nanoparticles can provide site specific drug delivery. The polymer used in Senna Nanoparticles formulations shows efficient and targeted drug release as well as reduction in dose, dosing frequency and side effects

Editorial Overview: Nanoscale Electrochemistry

A central challenge in electrochemical sciences is that the electrochemical response of an electrode is dominated by nanoscale features on the surface, yet our traditional electrochemical techniques operate on a millimeter or greater length scales. For instance, when we make a cyclic voltammetry measurement on a millimeter-scale electrode, the signal we obtain is based on the average response of all the active sites across the surface while details such as the activities of each site, their spatial distribution, and dynamics cannot be revealed. Nanoscale electrochemistry raises this challenge and has developed a range of techniques to effectively “zoom in” to the micro or nanoscale and, ultimately, to single molecules and atoms, enabling precise measurement of dynamic electrochemical process. This special edition highlights the cutting edge of nanoscale electrochemical research, spanning nanoparticle structure-activity relationships to DNA sequencing and 3D printing. A mainstay of modern nanoscale electrochemistry is the scanning droplet approach known as scanning electrochemical microscopy (SECCM). SECCM simply and effectively restricts an electrochemical measurement to micro or nanoscale region of a large sample surface. In this special edition (Table 1), Schuhmann and co-workers use SECCM to investigate the structure-activity relationships in a high entropy alloy and reveal that active site-specific activities can be detected with probes of dimensions below a micrometer.[1] Takahashi and coworkers use SECCM to investigate the capacitive response of carbon surfaces with 100-nanometer resolution and evaluate the difference in degradation of HOPG occurring at the edge and basal planes.[2] Caleb and co-workers apply a targeted electrochemical cell microscopy (TECCM) approach to isolate the electrocatalytic response of individual shape-controlled nanoparticles toward borohydride oxidation and reveal the significant variations in reactivity and stability for individual nanoparticles.[3] In the review by Bentley, the author summarizes how SECCM has been used to study (nano)particle electrochemistry, often isolated single nanoparticles dispersed on inert supports, and sometimes at sub-particles level.[4] Finally, Momotenko and coworkers review how scanning probe approaches, including but not limited to SECCM, can be utilized for micro and nanoscale electrochemical 3D printing, an innovative strategy for precise fabrication of micro and nanoscale structures.[5] A different approach of electrochemical measurements at nanointerfaces is nano-collision or nano-impact electrochemistry. Shen and Wang demonstrate three different configurations to investigate the size, surface charge, dielectric properties, and electrochemical features of individual graphene oxide sheets.[6] Another approach towards nanoscale electrochemistry is the advanced optical microscopy, where electrochemical processes at nanointerfaces can be imaged with high spatial and temporal resolution by detecting local optical properties. Willets and Bohn investigate the potential-dependent luminescence emission from three different electrofluorogenic probes an indium-tin oxide (ITO) surface.[7] A counterintuitive spectroelectrochemical observation at high irradiance or at low concentration is reported, highlighting the largely ignored importance of interaction between electrofluorogenic probes and ITO surfaces. Finally, we have two opinions that highlight how conductometric electrochemical sensors that make use of nanopores can effectively enable accurate measurements to single molecules. Tan and Ming review how biological nanopores can be used to detect nucleobase modifications in DNA,[8] while Johnson and co-workers elaborate how this approach can be used to sequence double-stranded DNA.[9] To summarize, nanoscale electrochemistry will continue to advance at space and time, revealing the intrinsic features that are often buried and gaining more complete understanding of intricate electrochemical process. Finally, we would like to express our thanks to the Publisher Dr. Brian P. Johnson and Editorial Manager Dr. Jing Tang for their kind supports during the preparation of this special edition. The authors declare no conflict of interest.

Single-Atom Catalysts (SACs) for Photocatalytic CO2 Reduction with H2 O: Activity, Product Selectivity, Stability, and Surface Chemistry.

In recent years, single-atom catalysts (SACs) have attracted the interest of researchers owing to their suitability for various catalytic applications. For instance, their optoelectronic features, site-specific activity, and cost-effectiveness make SACs ideal for photocatalytic CO2 reduction. The activity, product selectivity, and photostability of SACs depend on various factors such as the nature of the metal/support material, the interaction between the metal atoms and support, light-harvesting ability, charge separation behavior, CO2 adsorption ability, active sites, and defects. Consequently, it is necessary to investigate these factors in depth to elucidate the working principle(s) of SACs for catalytic applications. Herein, the recent progress in the development of SACs for photocatalytic CO2 reduction with H2 O is reviewed. First, a brief overview of CO2 photoreduction and SACs for CO2 conversion is provided. Several synthesis strategies and useful techniques for characterizing SACs employed in heterogeneous catalysis are then described. Next, the challenges of SACs for photocatalytic CO2 reduction and related optimization strategies, in terms of activity, product selectivity, and stability, are explored. The progress in the development of noble metal- and transition metal-based SACs and dual-SACs for photocatalytic CO2 reduction is discussed. Finally, the prospects of SACs for CO2 reduction are considered.

Regulation and Function of Metal Uptake Transporter NtNRAMP3 in Tobacco.

Natural resistance-associated macrophage protein (NRAMP) genes encode proteins with low substrate specificity, important for maintaining metal cross homeostasis in the cell. The role of these proteins in tobacco, an important crop plant with wide application in the tobacco industry as well as in phytoremediation of metal-contaminated soils, remains unknown. Here, we identified NtNRAMP3, the closest homologue to NRAMP3 proteins from other plant species, and functionally characterized it. A NtNRAMP3-GFP fusion protein was localized to the plasma membrane in tobacco epidermal cells. Expression of NtNRAMP3 in yeast was able to rescue the growth of Fe and Mn uptake defective Δfet3fet4 and Δsmf1 mutant yeast strains, respectively. Furthermore, NtNRAMP3 expression in wild-type Saccharomyces cerevisiae DY1457 yeast strain increased sensitivity to elevated concentrations of iron (Fe), manganese (Mn), copper (Cu), cobalt (Co), nickel (Ni), and cadmium (Cd). Taken together, these results point to a possible role in the uptake of metals. NtNRAMP3 was expressed in the leaves and to a lesser extent in the roots of tobacco plants. Its expression occurred mainly under control conditions and decreased very sharply in deficiency and excess of the tested metals. GUS-based analysis of the site-specific activity of the NtNRAMP3 promoter showed that it was primarily expressed in the xylem of leaf blades. Overall, our data indicate that the main function of NtNRAMP3 is to maintain cross homeostasis of Fe, Mn, Co, Cu, and Ni (also Cd) in leaves under control conditions by controlling xylem unloading.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

Polyvinyl Alcohol Cryogels for Acoustic Characterization of Phase-Change Contrast Agents.

Phase-change contrast agents (PCCAs) consisting of lipid-encapsulated low-boiling-point perfluorocarbons can be used in conjunction with ultrasound for diagnostic and therapeutic applications. One benefit of PCCAs is site-specific activation, whereby the liquid core is acoustically vaporized into a bubble detectable via ultrasound imaging. For further evaluation of PCCAs in a variety of applications, it is useful to disperse these nanodroplets into an acoustically compatible stationary matrix. However, many traditional phantom preparations require heating, which causes premature thermal activation of low-boiling-point PCCAs. Polyvinyl alcohol (PVA) cryogels do not require heat to set. Here we propose a simple method for the incorporation of the low-boiling-point PCCAs using octafluoropropane (OFP) and decafluorobutane (DFB) into PVA cryogels for a variety of acoustic characterization applications. We determined the utility of the phantoms by activating droplets with a focused transducer, visualizing the lesions with ultrasound imaging. At 1 MHz, droplet activation was consistently observed at 2.0 and 4.0 MPa for OFP and DFB, respectively.

Molecular basis of specificity and deamidation of eIF4A by Burkholderia Lethal Factor 1

Burkholderiapseudomallei lethal factor 1 (BLF1) exhibits site-specific glutamine deamidase activity against the eukaryotic RNA helicase, eIF4A, thereby blocking mammalian protein synthesis. The structure of a complex between BLF1 C94S and human eIF4A shows that the toxin binds in the cleft between the two RecA-like eIF4A domains forming interactions with residues from both and with the scissile amide of the target glutamine, Gln339, adjacent to the toxin active site. The RecA-like domains adopt a radically twisted orientation compared to other eIF4A structures and the nature and position of conserved residues suggests this may represent a conformation associated with RNA binding. Comparison of the catalytic site of BLF1 with other deamidases and cysteine proteases reveals that they fall into two classes, related by pseudosymmetry, that present either the re or si faces of the target amide/peptide to the nucleophilic sulfur, highlighting constraints in the convergent evolution of their Cys-His active sites.

Slow liquid dynamics near solid surfaces: Insights from site-resolved studies of ionic liquids in silica confinement.

We perform molecular dynamics simulations of ionic liquids that are confined between amorphous silica surfaces and composed of 1-butyl-3-methylimidazolium [C4mim] cations and tetrafluoroborate [BF4], hexafluorophosphate [PF6], or bis(trifluoromethylsulfonyl)imide [TFSI] anions. Near the silica surfaces, we observe strong slowdowns of ion dynamics, which involve mainly two layers of ions and amount to about two orders of magnitude, depending on temperature and anion type. For a detailed investigation of the slow interfacial dynamics, we determine the adsorption sites of the various anion species on the amorphous silica surfaces and ascertain the repopulation dynamics of these sites. The analysis reveals that the mean residence times show a broad distribution, where anions stay longer at sites, which provide better opportunities for hydrogen bonding. Furthermore, the mean residence times follow Arrhenius laws, providing access to site-specific activation energies Ei. The distributions G(Ei) have Gaussian shape with mean values from ∼0.40eV for TFSI to ∼0.48eV for PF6 and standard deviations of about 0.31eV. Thus, the amorphous silica surfaces impose static and disordered energy landscapes to the neighboring liquid, which have considerable ruggedness and, in this way, substantially hinder ion rearrangements. We discuss that qualitatively similar situations are expected for all kinds of confined liquids.

Use of passive acoustic monitoring to estimate fishing effort on artificial reefs in Alabama during the recreational red snapper fishing season

Estimating fishing effort is an important aspect of effective fisheries management for populations such as red snapper (Lutjanus campechanus) in the Gulf of Mexico. Monitoring effort, however, can be limited by where and when anglers can be easily observed, such as boat launches or aerial surveys. Passive acoustic monitoring (PAM) can be used to detect boat presence spatially and temporally, which can be used to infer fishing effort. In this study, we deployed PAM devices at multiple artificial reef sites (up to three at a time) in federal waters of the Alabama Reef Permit Zone during the recreational red snapper fishery in 2017 and 2018. Reefs at our deployment sites included multiple structure types (concrete and steel pyramid modules, bridge rubble reefs, chicken coops, and M1 tanks). Reef sites included a mix of publicly available reef coordinates (published reefs) and unpublished sites. To improve reliability of estimates of site-specific fishing activity from captured boat noise, we developed a method to automatically detect sounds indicative of idling vessels maintaining station (live-boating) near an artificial reef. Detections of boat gearshift sounds were consistent with our prediction that fishing effort would be reduced on unpublished sites, decrease as the season progressed, and show a strongly diurnal pattern. Counter to our prediction, fishing effort as measured by boat detections did not appear to differ between open and closed recreational fishing days at our sites, which may be partially explained by commercial fishers that operate on an individual quota system that allows for fishing on days closed to recreational fishers. Our results indicate that PAM in conjunction with this novel method could be an effective way to monitor daily and longer-term patterns of live-boating fishing vessel presence at specific artificial sites for the red snapper fishery in the Gulf of Mexico.

Best practices for predictions of radionuclide activity concentrations and total absorbed dose rates to freshwater organisms exposed to uranium mining/milling

Predictions of radionuclide dose rates to freshwater organisms can be used to evaluate the radiological environmental impacts of releases from uranium mining and milling projects. These predictions help inform decisions on the implementation of mitigation measures. The objective of this study was to identify how dose rate modelling could be improved to reduce uncertainty in predictions to non-human biota. For this purpose, we modelled the activity concentrations of 210Pb, 210Po, 226Ra, 230Th, and 238U downstream of uranium mines and mills in northern Saskatchewan, Canada, together with associated weighted absorbed dose rates for a freshwater food chain using measured activity concentrations in water and sediments. Differences in predictions of radionuclide activity concentrations occurred mainly from the different default partition coefficient and concentration ratio values from one model to another and including all or only some 238U decay daughters in the dose rate assessments. Consequently, we recommend a standardized best-practice approach to calculate weighted absorbed dose rates to freshwater biota whether a facility is at the planning, operating or decommissioned stage. At the initial planning stage, the best-practice approach recommend using conservative site-specific baseline activity concentrations in water, sediments and organisms and predict conservative incremental activity concentrations in these media by selecting concentration ratios based on species similarity and similar water quality conditions to reduce the uncertainty in dose rate calculations. At the operating and decommissioned stages, the best-practice approach recommends relying on measured activity concentrations in water, sediment, fish tissue and whole-body of small organisms to further reduce uncertainty in dose rate estimates. This approach would allow for more realistic but still conservative dose assessments when evaluating impacts from uranium mining projects and making decision on adequate controls of releases.

G3bp1 – microRNA-1 axis regulates cardiomyocyte hypertrophy

Adaptation of gene expression is one of the most fundamental response of cardiomyocytes to hypertrophic stimuli. G3bp1, an RNA binding protein with site-specific endoribonuclease activity regulates the processing of pre-miR-1 stem-loop, and thus levels of cardiomyocyte -enriched mature miR-1. Here, we examine the role of G3bp1 in regulating gene expression in quiescent cardiomyocytes and those undergoing growth-factor induced hypertrophy. Further, we determine if these changes are facilitated through G3bp1-mediated regulation of miR-1 in these cardiomyocytes. Using isolated cardiomyocytes with knockdown of endogenous G3bp1, we performed high throughput RNA sequencing to determine the change in cardiac transcriptome. Then, using gain and loss of function approach for both, G3bp1 and miR-1, alone or in combination we examine the G3bp1-miR-1 signaling in regulating gene expression and Endothelin (ET-1) -induced cardiomyocyte hypertrophy. We show that knockdown of endogenous G3bp1 results in inhibition of genes involved in calcium handling, cardiac muscle contraction, action potential and sarcomeric structure. In addition, there is inhibition of genes that contribute to hypertrophic and dilated cardiomyopathy development. Conversely, an increase is seen in genes that negatively regulate the Hippo signaling, like Rassf1 and Arrdc3, along with inflammatory genes of TGF-β and TNF pathways. Knockdown of G3bp1 restricts ET-1 induced cardiomyocyte hypertrophy. Interestingly, concurrent silencing of G3bp1 and miR-1 rescues the change in gene expression and inhibition of hypertrophy seen with knockdown of G3bp1 alone. Similarly, expression of exogenous G3bp1 reverses the miR-1 induced inhibition of gene expression. Intriguingly, expression of Gfp tagged G3bp1 results in perinuclear accumulations of G3bp1-Gfp, resembling Stress Granules. Based on our results, we conclude that G3bp1 through its regulation of mature miR-1 levels plays a critical role in regulating the expression of essential cardiac-enriched genes and those involved in development of cardiomyocyte hypertrophy.

Nanostructure system: Liposome – A bioactive carrier in drug delivery systems

The need for a new carrier system is always required to deliver the active drug content at the preferred target site in a predefined manner throughout the treatment period, because conventional dosage forms cannot achieve this desired effect. To overcome difficulties problems associated with contemporary drug formulations, nano formulations as nanocarriers with target-specific and site-specific activities are preferred. Nanocarrier liposomes consist of lipid bilayers encapsulating an aqueous core. In this study, liposomes are a new carrier system to deliver the encapsulated diagnostic and therapeutic agents in a specified manner and have led to significant interest in novel drug delivery systems as bioactive carriers. They can be considered “smart” bioactive carriers because of the presence of hydrophilic and lipophilic groups. It involves incorporating and protecting materials of different solubilities so that they can be delivered inside the human body and outside it, in vitro. By stabilising substances, removing barriers to cellular and tissue absorption, enhancing medication bio-distribution to target areas in vivo, and lowering systemic toxicity, they have increased the therapeutic efficacy of pharmaceuticals. The present review endeavours to highlight the potential development of liposomes as a new nanostructured carrier structure for drug delivery and preparation methods. This focuses on the method of preparation, lipososme nanoparticle for delivery of ion channel and way to improve the delivery of mRNA.