Considerations and recommendations for assessment of plasma protein binding and drug-drug interactions for siRNA therapeutics.

At the time of writing, although siRNA therapeutics are approved for human use, no official regulatory guidance specific to this modality is available. In the absence of guidance, preclinical development for siRNA followed a hybrid of the small molecule and biologics guidance documents. However, siRNA differs significantly from small molecules and protein-based biologics in its physicochemical, absorption, distribution, metabolism and excretion properties, and its mechanism of action. Consequently, certain reports typically included in filing packages for small molecule or biologics may benefit from adaption, or even omission, from an siRNA filing. In this white paper, members of the 'siRNA working group' in the IQ Consortium compile a list of reports included in approved siRNA filing packages and discuss the relevance of two in vitro reports-the plasma protein binding evaluation and the drug-drug interaction risk assessment-to support siRNA regulatory filings. Publicly available siRNA approval packages and the literature were systematically reviewed to examine the role of siRNA plasma protein binding and drug-drug interactions in understanding pharmacokinetic/pharmacodynamic relationships, safety and translation. The findings are summarized into two decision trees to help guide industry decide when in vitro siRNA plasma protein binding and drug-drug interaction studies are warranted.

Absorption, Distribution, Metabolism, and Excretion of [14C]-Sotorasib in Rats and Dogs: Interspecies Differences in Absorption, Protein Conjugation and Metabolism.

Sotorasib is a first-in-class, targeted covalent inhibitor of Kirsten rat sarcoma viral oncogene homolog (KRAS)G12C approved by the FDA to treat patients with locally advanced or metastatic non-small cell lung cancer with the KRASG12C mutation. The mass balance, excretion, and metabolism of [14C]-sotorasib was characterized in rats and dogs after a single dose of 60 or 500 mg/kg, respectively. Mean recovery was >90% for both species. Excretion of unchanged sotorasib was a minor pathway in rats, accounting for <4% of administered dose in urine and <7% of administered dose in feces. Approximately 66% of administered dose was recovered in the bile from bile duct cannulated rats as metabolites. Excretion of unchanged sotorasib was the major excretion pathway in dogs, likely caused by solubility-limited absorption. Major pathways of sotorasib biotransformation included glutathione conjugation and oxidative metabolism. In vitro experiments demonstrated that nonenzymatic conjugation (Michael addition) was the primary mechanism of the reaction with glutathione. Extended radioactivity profiles in blood and plasma were observed in rats, but not dogs, after dosing with [14C]-sotorasib. In vitro experiments demonstrated that sotorasib-protein adducts were observed with both rat hemoglobin and serum albumin, explaining the extended radioactivity profile. SIGNIFICANCE STATEMENT: This study characterized the mass balance, excretion, and metabolism of [14C]-sotorasib, a covalent Kirsten rat sarcoma viral oncogene homolog G12C inhibitor, in rats and dogs. Rapid absorption and extensive metabolism of sotorasib was observed in rats, while sotorasib was primarily excreted unchanged in dog feces, likely due to solubility-limited absorption. Protein adducts with rat hemoglobin and serum albumin were characterized, explaining observed extended blood and plasma radioactivity profiles. The primary biotransformation pathway, glutathione conjugation, was mediated through nonenzymatic conjugation.

A systematized review and qualitative synthesis of potential risk factors associated with the occurrence of non-O157 Shiga toxin-producing Escherichia coli (STEC) in the primary production of cattle.

Human infection with Shiga toxin-producing Escherichia coli (STEC) causes an estimated 2.8 million cases of acute illness worldwide each year. Serogroup O157 is the most commonly diagnosed STEC in humans, but cases linked to non-O157 STEC serogroups have increased recently due to increased surveillance and improvements to detection methods. Cattle are an important reservoir for STEC O157 and the same may be true for non-O157 STEC; therefore, reducing the occurrence of these pathogens in cattle could mitigate human infection risk. A systematized literature review of articles published within the Scopus database since 2010 (employing a partially systematic approach) was therefore conducted followed by qualitative synthesis of evidence to provide a structured overview of potential risk factors for non-O157 STEC in primary cattle production. Overall, few relevant studies were identified (n = 22), highlighting that more studies are needed. Consistently significant associations were only identified with respect to cattle age (broadly higher rate of isolation from young animals compared to adults) and season of sampling (generally increased isolation of non-O157 STEC in summer). However, wide variation in study designs, including notable differences in laboratory detection methods, means drawing more general conclusions is currently not possible based on the results of this review. However, it is likely that the development of more sensitive methods for non-O157 STEC detection in potential livestock reservoirs and increased standardization across statistically sound epidemiological investigations are required to identify pertinent risk factors.

Laboratory-scale evaluation of algaecide effectiveness for control of microcystin-producing cyanobacteria from Lake Okeechobee, Florida (USA).

Growth of microcystin-producing cyanobacteria in Lake Okeechobee (Florida, USA) and surrounding waters has resulted in adverse health impacts for humans and endangered species, as well as significant economic losses. As these issues worsen, there is growing pressure for efficacious solutions to rapidly mitigate harmful algal blooms (HABs) and protect critical freshwater resources. Applications of USEPA-registered algaecides as management tactics meet many decision-making criteria often required by water resource managers (e.g., effective, scalable, selective), but have not yet been evaluated on a large scale within the Lake Okeechobee waterway. This study was conducted to bolster the peer-reviewed database for available management tactics against microcystin-producing cyanobacteria in waters of this region. Laboratory-scale experiments can be conducted first to minimize uncertainty at larger scales and improve confidence in decision-making. In this study, samples containing microcystin-producing cyanobacteria collected from Lake Okeechobee were exposed to several USEPA-registered algaecides in laboratory toxicity experiments. Responses of target cyanobacteria were measured 3 days after treatment (DAT) in terms of cell density, chlorophyll-a concentrations, and phycocyanin concentrations. Based on responses of the cyanobacteria, minimum effective exposure concentrations were identified for each algaecide. Microcystin release (i.e. proportion of total microcystins in the aqueous phase) was measured and compared 1 DAT among effective exposures. Total microcystin concentrations were measured in effective treatments at 1, 4, and 9 DAT to discern potential for microcystin persistence following exposures to the effective formulations and exposure concentrations. Overall, several formulations including GreenClean Liquid® 5.0, GreenClean Liquid® 5.0 combined with Hydrothol® 191, and the copper-based algaecides evaluated (Algimycin® PWF, Argos, Captain® XTR, Cutrine® Ultra, and SeClear®) achieved significant and similar effects on target cyanobacteria. The chelated copper-based formulations (Algimycin® PWF, Argos, Captain® XTR, and Cutrine® Ultra) resulted in relatively less microcystin release 1 DAT and lesser total microcystin concentrations 4 DAT. At 9 DAT, total microcystin concentrations were significantly lower than in untreated controls in all treatments evaluated. These results provide the necessary comparative performance data for preliminary decision-making and designing additional studies at larger scales. Importantly, the comparative toxicity data and approach provided in this study demonstrate the initial steps for development of site-specific management strategies for Lake Okeechobee and other areas impacted by harmful algal blooms with large spatial and temporal scales.

Numerical Analysis of Time-Dependent Inhibition by MDMA.

Methylenedioxymethamphetamine (MDMA) is a known drug of abuse and schedule 1 narcotic under the Controlled Substances Act. Previous pharmacokinetic work on MDMA used classic linearization techniques to conclude irreversible mechanism-based inhibition of CYP2D6. The current work challenges this outcome by assessing the possibility of two alternative reversible kinetic inhibition mechanisms known as the quasi-irreversible (QI) model and equilibrium model (EM). In addition, progress curve experiments were used to investigate the residual metabolism of MDMA by liver microsomes and CYP2D6 baculosomes over incubation periods up to 30 minutes. These experiments revealed activity in a terminal linear phase at the fractional rates with respect to initial turnover of 0.0354 ± 0.0089 in human liver microsomes and 0.0114 ± 0.0025 in baculosomes. Numerical model fits to percentage of remaining activity (PRA) data were consistent with progress curve modeling results, wherein an irreversible inhibition pathway was found unnecessary for good fit scoring. Both QI and EM kinetic mechanisms fit the PRA data well, although in CYP2D6 baculosomes the inclusion of an irreversible inactivation pathway did not allow for convergence to a reasonable fit. The kinetic complexity accessible to numerical modeling has been used to determine that MDMA is not an irreversible inactivator of CYP2D6, and instead follows what can be generally referred to as slowly reversible inhibition. SIGNIFICANCE STATEMENT: The work herein describes the usage of computational models to delineate between irreversible and slowly reversible time-dependent inhibition. Such models are used in the paper to analyze MDMA and classify it as a reversible time-dependent inhibitor.

Time Course of Aldehyde Oxidase and Why It Is Nonlinear.

Many promising drug candidates metabolized by aldehyde oxidase (AOX) fail during clinical trial owing to underestimation of their clearance. AOX is species-specific, which makes traditional allometric studies a poor choice for estimating human clearance. Other studies have suggested using half-life calculated by measuring substrate depletion to measure clearance. In this study, we proposed using numerical fitting to enzymatic pathways other than Michaelis-Menten (MM) to avoid missing the initial high turnover rate of product formation. Here, product formation over a 240-minute time course of six AOX substrates-O6-benzylguanine, N-(2-dimethylamino)ethyl)acridine-4-carboxamide, zaleplon, phthalazine, BIBX1382 [N8-(3-Chloro-4-fluorophenyl)-N2-(1-methyl-4-piperidinyl)-pyrimido[5,4-d]pyrimidine-2,8-diamine dihydrochloride], and zoniporide-have been provided to illustrate enzyme deactivation over time to help better understand why MM kinetics sometimes leads to underestimation of rate constants. Based on the data provided in this article, the total velocity for substrates becomes slower than the initial velocity by 3.1-, 6.5-, 2.9-, 32.2-, 2.7-, and 0.2-fold, respectively, in human expressed purified enzyme, whereas the K m remains constant. Also, our studies on the role of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, show that ROS did not significantly alter the change in enzyme activity over time. Providing a new electron acceptor, 5-nitroquinoline, did, however, alter the change in rate over time for mumerous compounds. The data also illustrate the difficulties in using substrate disappearance to estimate intrinsic clearance.

Field Interventions Against Colonization of Broilers by Campylobacter.

Poultry accounts for a high proportion of human campylobacteriosis cases, and the problem of Campylobacter colonization of broiler flocks has proven to be intractable. Owing to their broad host range and genetic instability, Campylobacter organisms are ubiquitous and adaptable in the broiler farm environment, colonizing birds heavily and spreading rapidly after introduction into a flock. This review examines strategies to prevent or suppress such colonization, with a heavy emphasis on field investigations. Attempts to exclude Campylobacter via enhanced biosecurity and hygiene measures have met with mixed success. Reasons for this are becoming better understood as investigations focus on houses, ventilation, biosecurity practices, external operators, and compliance, among other factors. It is evident that piecemeal approaches are likely to fail. Complementary measures include feed and drinking water treatments applied in either preventive or suppressive modes using agents including organic acids and their derivatives, also litter treatments, probiotics, prebiotics, and alterations to diet. Some treatments aim to reduce the number of Campylobacter organisms entering abattoirs by suppressing intestinal colonization just before slaughter; these include acid water treatment or administration of bacteriophages or bacteriocins. Experimental vaccines historically have had little success, but some recent subunit vaccines show promise. Overall, there is wide variation in the control achieved, and consistency and harmonization of trials is needed to enable robust evaluation. There is also some potential to breed for resistance to Campylobacter. Good and consistent control of flock colonization by Campylobacter may require an as-yet undetermined combination of excellent biosecurity plus complementary measures.

Implementation of a Risk-Stratified Opioid and Benzodiazepine Weaning Protocol in a Pediatric Cardiac ICU.

OBJECTIVES: Opioids and benzodiazepines are commonly used to provide analgesia and sedation for critically ill children with cardiac disease. These medications have been associated with adverse effects including delirium, dependence, withdrawal, bowel dysfunction, and potential neurodevelopmental abnormalities. Our objective was to implement a risk-stratified opioid and benzodiazepine weaning protocol to reduce the exposure to opioids and benzodiazepines in pediatric patients with cardiac disease. DESIGN: A prospective pre- and postinterventional study. PATIENTS: Critically ill patients less than or equal to 21 years old with acquired or congenital cardiac disease exposed to greater than or equal to 7 days of scheduled opioids ± scheduled benzodiazepines between January 2013 and February 2015. SETTING: A 24-bed pediatric cardiac ICU and 21-bed cardiovascular acute ward of an urban stand-alone children's hospital. INTERVENTION: We implemented an evidence-based opioid and benzodiazepine weaning protocol using educational and quality improvement methodology. MEASUREMENTS AND MAIN RESULTS: One-hundred nineteen critically ill children met the inclusion criteria (64 post intervention, 55 pre intervention). Demographics and risk factors did not differ between groups. Patients in the postintervention period had shorter duration of opioids (19.0 vs 30.0 d; p < 0.01) and duration of benzodiazepines (5.3 vs 22.7 d; p < 0.01). Despite the shorter duration of wean, there was a decrease in withdrawal occurrence (% Withdrawal Assessment Tool score ≥ 4, 4.9% vs 14.1%; p < 0.01). There was an 8-day reduction in hospital length of stay (34 vs 42 d; p < 0.01). There was a decrease in clonidine use (14% vs 32%; p = 0.02) and no change in dexmedetomidine exposure (59% vs 75%; p = 0.08) in the postintervention period. CONCLUSIONS: We implemented a risk-stratified opioid and benzodiazepine weaning protocol for critically ill cardiac children that resulted in reduction in opioid and benzodiazepine duration and dose exposure, a decrease in symptoms of withdrawal, and a reduction in hospital length of stay.

Toward a systems approach to the human cytochrome P450 ensemble: interactions between CYP2D6 and CYP2E1 and their functional consequences.

Functional cross-talk among human drug-metabolizing cytochrome P450 through their association is a topic of emerging importance. Here, we studied the interactions of human CYP2D6, a major metabolizer of psychoactive drugs, with one of the most prevalent human P450 enzymes, ethanol-inducible CYP2E1. Detection of P450-P450 interactions was accomplished through luminescence resonance energy transfer between labeled proteins incorporated into human liver microsomes and the microsomes of insect cells containing NADPH-cytochrome P450 reductase. The potential of CYP2D6 to form oligomers in the microsomal membrane is among the highest observed with human cytochrome P450 studied up to date. We also observed the formation of heteromeric complexes of CYP2D6 with CYP2E1 and CYP3A4, and found a significant modulation of these interactions by 3,4-methylenedioxymethylamphetamine, a widespread drug of abuse metabolized by CYP2D6. Our results demonstrate an ample alteration of the catalytic properties of CYP2D6 and CYP2E1 caused by their association. In particular, we demonstrated that preincubation of microsomes containing co-incorporated CYP2D6 and CYP2E1 with CYP2D6-specific substrates resulted in considerable time-dependent activation of CYP2D6, which presumably occurs via a slow substrate-induced reorganization of CYP2E1-CYP2D6 hetero-oligomers. Furthermore, we demonstrated that the formation of heteromeric complexes between CYP2E1 and CYP2D6 affects the stoichiometry of futile cycling and substrate oxidation by CYP2D6 by means of decreasing the electron leakage through the peroxide-generating pathways. Our results further emphasize the role of P450-P450 interactions in regulatory cross-talk in human drug-metabolizing ensemble and suggest a role of interactions of CYP2E1 with CYP2D6 in pharmacologically important instances of alcohol-drug interactions.

Relationship among aqueous copper half-lives and responses of Pimephales promelas to a series of copper sulfate pentahydrate concentrations.

Copper algaecide exposures in situ are often of shorter duration than exposures for static toxicity experiments because aqueous concentrations in situ dissipate as a function of site-specific fate processes. Consequently, responses of organisms to static copper exposures may overestimate effects following in situ exposures. To understand the role of exposure duration for altering responses, Pimephales promelas survival was compared following static (96 h) and pulse (1.5, 4, 8, and 15 h half-lives) exposures of CuSO4•5H2O. Copper concentrations sorbed by fry indicated a consequence of different exposures. Responses of P. promelas to static exposures resulted in 96 h LC50s of 166 µgCu/L (95% confidence interval [CI], 142-189 µgCu/L) as soluble copper and 162 µgCu/L (CI, 140-183 µgCu/L) as acid soluble copper. Relative to static 96 h LC50s, exposures with half-lives of 1.5, 4 and 8 h resulted in LC50s 10, 3 and 2 times greater, respectively, for responses measured 96 h after exposure initiation. Copper concentrations extracted from fry exposed for 1.5, 4 and 8 h half-lives were less than the static experiment. However, copper sorbed by fry in the 15 h half-life experiment was not different than the static experiment. The relationship between 96 h LC50 and 1/half-life was expressed using the equations y = 116 + 1360 × (R2 = 0.97) for soluble copper and y = 147 + 1620 × (R2 = 0.98) for acid soluble copper. Incorporation of exposure duration for predictions of P. promelas responses to copper pulse exposures increases prediction accuracy by an order of magnitude.

Cell density dependence of Microcystis aeruginosa responses to copper algaecide concentrations: Implications for microcystin-LR release.

Along with mechanistic models, predictions of exposure-response relationships for copper are often derived from laboratory toxicity experiments with standardized experimental exposures and conditions. For predictions of copper toxicity to algae, cell density is a critical factor often overlooked. For pulse exposures of copper-based algaecides in aquatic systems, cell density can significantly influence copper sorbed by the algal population, and consequent responses. A cyanobacterium, Microcystis aeruginosa, was exposed to a copper-based algaecide over a range of cell densities to model the density-dependence of exposures, and effects on microcystin-LR (MC-LR) release. Copper exposure concentrations were arrayed to result in a gradient of MC-LR release, and masses of copper sorbed to algal populations were measured following exposures. While copper exposure concentrations eliciting comparable MC-LR release ranged an order of magnitude (24-h EC50s 0.03-0.3mg Cu/L) among cell densities of 106 through 107 cells/mL, copper doses (mg Cu/mg algae) were similar (24-h EC50s 0.005-0.006mg Cu/mg algae). Comparisons of MC-LR release as a function of copper exposure concentrations and doses provided a metric of the density dependence of algal responses in the context of copper-based algaecide applications. Combined with estimates of other site-specific factors (e.g. water characteristics) and fate processes (e.g. dilution and dispersion, sorption to organic matter and sediments), measuring exposure-response relationships for specific cell densities can refine predictions for in situ exposures and algal responses. These measurements can in turn decrease the likelihood of amending unnecessary copper concentrations to aquatic systems, and minimize risks for non-target aquatic organisms.

Comparative toxicity of sodium carbonate peroxyhydrate to freshwater organisms.

Sodium carbonate peroxyhydrate (SCP) is a granular algaecide containing H2O2 as an active ingredient to control growth of noxious algae. Measurements of sensitivities of target and non-target species to hydrogen peroxide are necessary for water resource managers to make informed decisions and minimize risks for non-target species when treating noxious algae. The objective of this study was to measure and compare responses among a target noxious alga (cyanobacterium Microcystis aeruginosa) and non-target organisms including a eukaryotic alga (chlorophyte Pseudokirchneriella subcapitata), microcrustacean (Ceriodaphnia dubia), benthic amphipod (Hyalella azteca), and fathead minnow (Pimephales promelas) to exposures of hydrogen peroxide as SCP. Hydrogen peroxide exposures were confirmed using the I3(-) method. SCP margins of safety for these organisms were compared with published toxicity data to provide context for other commonly used algaecides and herbicides (e.g. copper formulations, endothall, and diquat dibromide). Algal responses (cell density and chlorophyll a concentrations) and animal mortality were measured after 96h aqueous exposures to SCP in laboratory-formulated water to estimate EC50 and LC50 values, as well as potency slopes. Despite a shorter test duration, M. aeruginosa was more sensitive to hydrogen peroxide as SCP (96h EC50:0.9-1.0mgL(-)(1) H2O2) than the eukaryotic alga P. subcapitata (7-d EC50:5.2-9.2mgL(-1) H2O2), indicating potential for selective control of prokaryotic algae. For the three non-target animals evaluated, measured 96-h LC50 values ranged from 1.0 to 19.7mgL(-1) H2O2. C. dubia was the most sensitive species, and the least sensitive species was P. promelas, which is not likely to be affected by concentrations of hydrogen peroxide as SCP that would be used to control noxious algae (e.g. M. aeruginosa). Based on information from peer-reviewed literature, other algaecides could be similarly selective for cyanobacteria. Of the algaecides compared, SCP can selectively mitigate risks associated with noxious cyanobacterial growths (e.g. M. aeruginosa), with an enhanced margin of safety for non-target species (e.g. P. promelas).

Influence of commercial (Fluka) naphthenic acids on acid volatile sulfide (AVS) production and divalent metal precipitation.

Energy-derived waters containing naphthenic acids (NAs) are complex mixtures often comprising a suite of potentially problematic constituents (e.g. organics, metals, and metalloids) that need treatment prior to beneficial use, including release to receiving aquatic systems. It has previously been suggested that NAs can have biostatic or biocidal properties that could inhibit microbially driven processes (e.g. dissimilatory sulfate reduction) used to transfer or transform metals in passive treatment systems (i.e. constructed wetlands). The overall objective of this study was to measure the effects of a commercially available (Fluka) NA on sulfate-reducing bacteria (SRB), production of sulfides (as acid-volatile sulfides [AVS]), and precipitation of divalent metals (i.e. Cu, Ni, Zn). These endpoints were assessed following 21-d aqueous exposures of NAs using bench-scale reactors. After 21-days, AVS molar concentrations were not statistically different (p<0.0001; α=0.05) among NA treatments (10, 20, 40, 60, and 80mg NA/L) and an untreated control (no NAs). Extent of AVS production was sufficient in all NA treatments to achieve ∑SEM:AVS <1, indicating that conditions were conducive for treatment of metals, with sulfide ligands in excess of SEM (Cu, Ni, and Zn). In addition, no adverse effects to SRB (in terms of density, relative abundance, and diversity) were measured following exposures of a commercial NA. In this bench-scale study, dissimilatory sulfate reduction and subsequent metal precipitation were not vulnerable to NAs, indicating passive treatment systems utilizing sulfide production (AVS) could be used to treat metals occurring in NAs affected waters.

MHC class Ib molecules bridge innate and acquired immunity.

Our understanding of the classical MHC class I molecules (MHC class Ia molecules) has long focused on their extreme polymorphism. These molecules present peptides to T cells and are central to discrimination between self and non-self. By contrast, the functions of the non-polymorphic MHC class I molecules (MHC class Ib molecules) have been elusive, but emerging evidence reveals that, in addition to antigen presentation, MHC class Ib molecules are involved in immunoregulation. As we discuss here, the subset of MHC class Ib molecules that presents peptides to T cells bridges innate and acquired immunity, and this provides insights into the origins of acquired immunity.

Evaluation of the utility of six measures for algal (Microcystis aeruginosa, Planktothrix agardhii and Pseudokirchneriella subcapitata) viability.

Standard algal toxicity tests are used to discern responses of algae to a variety of exposures including pesticides, personal care products and complex mixtures such as runoff and effluents. There are concerns regarding the accuracy, precision and utility of algal viability measures used as endpoints in algal toxicity test protocols. To definitively evaluate six algal viability measures, algae were heat-treated to produce known live:dead cell ratios. Cultures of two prokaryotic algae (Microcystis aeruginosa and Planktothrix agardhii) and a eukaryotic alga (Pseudokirchneriella subcapitata) were boiled for five minutes and mixed after cooling with untreated cultures to produce suspensions of 0%, 25%, 50%, 75% and 100% live algal cells. Optical microscopy was used to assess the viability of algae on a cell-by-cell basis by measuring cell density, uptake of a vital stain (neutral red) and exclusion of a mortal stain (erythrosin b). Aggregate measures of algal cell viability included chlorophyll a concentrations, pheophytin a concentrations and respiration (measured as 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium formazan absorbance (INT)). Cell densities, erythrosin b stained cells and chlorophyll a concentrations correlated with viable M. aeruginosa, P. agardhii and P. subcapitata cells (R(2)=0.97-0.78, 0.98-0.85 and 0.99-0.97 respectively). Pheophytin a concentrations and neutral red stained cells did not correlate with viable algae (R(2)=0.41-0.01 and 0.15-0.03 respectively). For INT formazan absorbance, 50%, 75% and 100% viable algae had greater variances and did not strongly correlate (R(2)=0.75-0.54). This result was likely confounded by respiration associated with resident bacteria. Three of the six methods provided accurate and precise information regarding the viability of both prokaryotic and eukaryotic algae. These methods also have a relatively low initial expense and can be used widely.

Responses of Lyngbya wollei to algaecide exposures and a risk characterization associated with their use.

To make informed decisions regarding management of noxious algal growths, water resource managers require information on responses of target and non-target species to algaecide exposures. Periodic treatments of Phycomycin®-SCP (sodium carbonate peroxyhydrate) followed by Algimycin®-PWF (gluconate and citrate chelated copper) to control Lyngbya wollei growths for ten years provided an opportunity for a risk evaluation of treated coves in Lay Lake, AL. Abiotic sediment characteristics (acid soluble copper concentrations, acid volatile sulfides, percent organic matter and cation exchange capacity) and survival of Hyalella azteca and Chironomus dilutus were measured in sediment samples from treated and untreated coves to assess the bioavailability of potential copper-residuals. In laboratory studies to seek a more effective approach for managing the growth of Lyngbya, six algaecide treatments consisting of combinations of copper-based algaecides (Cutrine®-Ultra, Clearigate® and Algimycin®- PWF), a hydrogen peroxide based algaecide (Phycomycin®-SCP) and an adjuvant (Cide-Kick II) were assessed for efficacy in controlling L. wollei sampled from Lay Lake. The most efficient algaecide treatment was determined based on post-treatment algal wet weight and visual observations of responses to exposures. To estimate the margin of safety for non-target organisms, Pimephales promelas was exposed to the most efficacious treatment and a treatment of Phycomycin®-SCP followed by Algimycin®-PWF. Results from sediment experiments demonstrated that there were no measureable copper residuals and no adverse effects on H. azteca and C. dilutus from sediments following ten years of copper-based algaecide treatments. Based on the laboratory results, a treatment of Phycomycin®-SCP at 10.1 mg H2O2/L followed by Cide-Kick II at 0.2 mg/L and Algimycin®- PWF at 0.26 mg Cu/L could control the growth of Lyngbya wollei from Lay Lake, AL and enhance the margin of safety for non-target species (e.g. P. promelas).

Novelty and anxiolytic drugs dissociate two components of hippocampal theta in behaving rats.

Hippocampal processing is strongly implicated in both spatial cognition and anxiety and is temporally organized by the theta rhythm. However, there has been little attempt to understand how each type of processing relates to the other in behaving animals, despite their common substrate. In freely moving rats, there is a broadly linear relationship between hippocampal theta frequency and running speed over the normal range of speeds used during foraging. A recent model predicts that spatial-translation-related and arousal/anxiety-related mechanisms of hippocampal theta generation underlie dissociable aspects of the theta frequency-running speed relationship (the slope and intercept, respectively). Here we provide the first confirmatory evidence: environmental novelty decreases slope, whereas anxiolytic drugs reduce intercept. Variation in slope predicted changes in spatial representation by CA1 place cells and novelty-responsive behavior. Variation in intercept predicted anxiety-like behavior. Our findings isolate and doubly dissociate two components of theta generation that operate in parallel in behaving animals and link them to anxiolytic drug action, novelty, and the metric for self-motion.

Prevalence, antibiotic resistance, and genomic characterisation of Campylobacter spp. in retail chicken in Hanoi, Vietnam.

Campylobacter spp. are a leading cause of bacterial foodborne zoonosis worldwide, with poultry meat and products recognised as a significant source of human infection. In Vietnam there are few data regarding the occurrence, antimicrobial resistance, and genomic diversity of Campylobacter in poultry and poultry meat. The aim of this study was to estimate the prevalence of Campylobacter in chicken meat at retail in Hanoi, determine antimicrobial sensitivities of the Campylobacter isolated, and assess their genetic diversity. A total of 120 chicken meat samples were collected from eight traditional retail markets (n=80) and four supermarkets (n=40). Campylobacter was isolated following ISO 10272-1 : 2017 and identification verified by PCR. The prevalence of Campylobacter was 38.3 % (46/120) and C. coli was the most prevalent species in both retail markets (74 %) and supermarkets (88 %). The minimum inhibitory concentrations for ciprofloxacin, erythromycin, gentamicin, nalidixic acid, streptomycin, and tetracycline were determined by broth microdilution for 32 isolates. All characterised Campylobacter were resistant to ciprofloxacin, nalidixic acid, and tetracycline, with corresponding resistance determinants detected in the sequenced genomes. Most C. coli were multidrug resistant (24/28) and two harboured the erythromycin resistance gene ermB on a multiple drug-resistance genomic island, a potential mechanism for dissemination of resistance. The 32 isolates belonged to clonal complexes associated with both poultry and people, such as CC828 for C. coli. These results contribute to the One Health approach for addressing Campylobacter in Vietnam by providing detailed new insights into a main source of human infection and can inform the design of future surveillance approaches.

Evidence for phenotypic plasticity among multihost Campylobacter jejuni and C. coli lineages, obtained using ribosomal multilocus sequence typing and Raman spectroscopy.

Closely related bacterial isolates can display divergent phenotypes. This can limit the usefulness of phylogenetic studies for understanding bacterial ecology and evolution. Here, we compare phenotyping based on Raman spectrometric analysis of cellular composition to phylogenetic classification by ribosomal multilocus sequence typing (rMLST) in 108 isolates of the zoonotic pathogens Campylobacter jejuni and C. coli. Automatic relevance determination (ARD) was used to identify informative peaks in the Raman spectra that could be used to distinguish strains in taxonomic and host source groups (species, clade, clonal complex, and isolate source/host). Phenotypic characterization based on Raman spectra showed a degree of agreement with genotypic classification using rMLST, with segregation accuracy between species (83.95%), clade (in C. coli, 98.41%), and, to some extent, clonal complex (86.89% C. jejuni ST-21 and ST-45 complexes) being achieved. This confirmed the utility of Raman spectroscopy for lineage classification and the correlation between genotypic and phenotypic classification. In parallel analysis, relatively distantly related isolates (different clonal complexes) were assigned the correct host origin irrespective of the clonal origin (74.07 to 96.97% accuracy) based upon different Raman peaks. This suggests that the phenotypic characteristics, from which the phenotypic signal is derived, are not fixed by clonal descent but are influenced by the host environment and change as strains move between hosts.

Activation of Wnt signaling arrests effector differentiation in human peripheral and cord blood-derived T lymphocytes.

The canonical Wnt/ß-catenin signaling pathway plays an important role in thymocyte development and T cell migration, but little is known about its role in naive-to-effector differentiation in human peripheral T cells. We show that activation of Wnt/ß-catenin signaling arrests human peripheral blood and cord blood T lymphocytes in the naive stage and blocks their transition into functional T effector cells. Wnt signaling was induced in polyclonally activated human T cells by treatment either with the glycogen synthase kinase 3ß inhibitor TWS119 or the physiological Wnt agonist Wnt-3a, and these T cells preserved a naive CD45RA(+)CD62L(+) phenotype compared with control-activated T cells that progressed to a CD45RO(+)CD62L(-) effector phenotype, and this occurred in a TWS119 dose-dependent manner. TWS119-induced Wnt signaling reduced T cell expansion, as a result of a block in cell division, and impaired acquisition of T cell effector function, measured by degranulation and IFN-γ production in response to T cell activation. The block in T cell division may be attributed to the reduced IL-2Rα expression in TWS119-treated T cells that lowers their capacity to use autocrine IL-2 for expansion. Collectively, our data suggest that Wnt/ß-catenin signaling is a negative regulator of naive-to-effector T cell differentiation in human T lymphocytes. The arrest in T cell differentiation induced by Wnt signaling might have relevant clinical applications such as to preserve the naive T cell compartment in Ag-specific T cells generated ex vivo for adoptive T cell immunotherapy.