Search

Purpose Dry eye syndrome is one of the most common reasons for eye-related discomfort which, without treatment, in some cases may even lead to corneal damage. Blinking, baseline and reflex lachrymation and drainage compromise the topical application of therapeutics demanding repeated, often hourly applications of common lubricants. In contrast, topically administered chitosan-N-acetylcysteine-based eye drops were reported to sustain on the ocular surface for more than 24 h. The thiolated biopolymer can interact with the corneal mucin layer thereby forming covalent disulphide bridges, which may contribute to extended residence times. Design/methodology/approach In this study, the tribological characteristics of four different lubricants including hyaluronic acid and chitosan-N-acetylcysteine containing commercially available eye drops were investigated. For this purpose, a representative test setup was developed, which mimics the contact between the cornea and the eyelid wiper. Gels with different elastic properties coated with a mucin layer were used as a substrate to mimic the corneal surface. Tests were conducted with a micro-tribometer, and friction values were recorded. Contact zones were characterized by X-ray photoelectron spectroscopy to investigate wear and thiol bonding on the surface. Findings Results revealed the lowest average coefficient of friction values for chitosan-N-acetylcysteine-based eye drops and substrate dependence of the test setup. Originality/value In this study, the authors introduced an in vitro system to test different types of eye drops so that chemical interaction with the mucin layer can be observed. These interactions change the tribological performance significantly and must be considered to have results relevant to the actual application.

We present obsidian-sourcing data from Pot Creek Pueblo (LA 260), one of the northernmost Puebloan settlements in the Northern Rio Grande, occupied from at least 1260 ce until ca. 1320 ce when much of the pueblo was burned and the site was depopulated. Although the occupation of Pot Creek Pueblo was short, it occurred during a pivotal period in the Northern Rio Grande. The population of the region increased rapidly at this time, possibly due to an influx of migrants from the Mesa Verde/San Juan area to the west, and locally people living in relatively small villages comprised of pithouses and above-ground unit pueblos begin to coalesce into fewer, but much larger above-ground pueblos. Obsidian-source choices throughout the region may provide insight into how the proposed migration impacted existing resource-procurement patterns. Our data demonstrate that despite the diverse histories of the settlement’s inhabitants, the exchange system supplying obsidian to Pot Creek Pueblo remained stable throughout its occupation. We argue that exchange dynamics of the local community remained the primary means of obtaining obsidian despite potential for new avenues that might have been available through the addition of new community members from outside the region and despite population growth and changing settlement patterns in the broader Northern Rio Grande.

Surfactants play an important role in stabilizing proteins in liquid formulations against aggregate/particle formation during processing, handling, storage, and transportation. Only 3 surfactants are currently used in marketed therapeutic protein formulations: polysorbate 20, polysorbate 80, and poloxamer 188. While polysorbates are the most widely used surfactants, their intrinsic oxidative and hydrolytic degradation issues highlights the importance of alternative surfactants such as poloxamer 188. Here, we compare polysorbates and poloxamer 188 with regards to their stabilizing properties under various stress and storage conditions for several monoclonal antibody formulations. Our data shows that poloxamer 188 can provide suitable protection of monoclonal antibodies against interfacial stress in liquid formulations in vials. However, visible protein-polydimethylsiloxane (PDMS; silicone oil) particles were observed in vials after long-term storage at 2-8°C for some protein formulations using poloxamer 188, which were not observed in polysorbate formulations. The occurrence of these protein-PDMS particles in poloxamer 188 formulations is a protein-specific phenomenon that may correlate with protein physico-chemical properties. In this study, the primary source of the PDMS in particles found in vials was considered to be from the primary packaging stoppers used. Our findings highlight benefits, but also risks associated with using poloxamer 188 in liquid biotherapeutic formulations.

The first goal of this study is to identify the ideal piezoelectric material for the manufacturing of rheological reflectance ultrasonic sensors. The second goal is to integrate the ultrasonic rheometer within a gas absorption reactor and to measure viscosity changes in an ionic liquid (IL) caused by gas absorption. To achieve the objectives, bismuth titanate, lead titanate, lead metaniobate and lead zirconate titanate materials in layer, tungsten bronze and perovskite structures were assembled on aluminum delay lines and tested under thermal cycling between room temperature and 150 &deg, C. The results showed that lead metaniobate in tungsten bronze structure is the most suitable material for long time duration thermal cycling. Therefore, the ultrasonic rheometer was assembled using this material and installed in a pressurized reactor to test a reference IL at the operating conditions of 50 &deg, C and at a pressure of 80 bar. The reference IL was saturated with nitrogen as well as hydrogen gas. Viscosity signals remained constant under the hydrogen atmosphere, while in nitrogen atmosphere the absorption of the gas lead to a rise in the value of viscosity.

The main objective of the MACIVIVA European consortium was to develop new Good Manufacturing Practice pilot lines for manufacturing thermostable vaccines with stabilized antigens on influenza virosomes as enveloped virus-like particles. The HIV-1 gp41-derived antigens anchored in the virosome membrane, along with the adjuvant 3M-052 (TLR7/8 agonist) on the same particle, served as a candidate vaccine for the proof of concept for establishing manufacturing processes, which can be directly applied or adapted to other virosomal vaccines or lipid-based particles. Heat spray-dried powders suitable for nasal or oral delivery, and freeze-dried sublingual tablets were successfully developed as solid dosage forms for mucosal vaccination. The antigenic properties of vaccinal antigens with key gp41 epitopes were maintained, preserving the original immunogenicity of the starting liquid form, and also when solid forms were exposed to high temperature (40 °C) for up to 3 months, with minimal antigen and adjuvant content variation. Virosomes reconstituted from the powder forms remained as free particles with similar size, virosome uptake by antigen-presenting cells in vitro was comparable to virosomes from the liquid form, and the presence of excipients specific to each solid form did not prevent virosome transport to the draining lymph nodes of immunized mice. Virosome integrity was also preserved during exposure to

Introduction This review summarizes the research conducted on botulinum toxin (BoTx) from 1943 to 1956 by a small group of Camp Detrick investigators and their staff. A systematic, cross-disciplinary approach was used to develop effective vaccines against this biological warfare threat agent. In response to the potential need for medical countermeasures against BoTx during World War II, the refinement of isolation and purification techniques for BoTx successfully led to the large-scale production of botulinum toxoid vaccines. In addition, the work at Camp Detrick provided the foundation for the subsequent use of BoTx as a tool for studying the trophic regulation of skeletal muscle within motor neuron terminals and, more recently, for elucidation of the intricate details of neurotransmitter release at the molecular level. Indirectly, Camp Detrick investigators also played a significant role in studies that culminated in the use of BoTx as a pharmaceutical product that has been approved by the U.S. Food and Drug Administration for treating movement disorders, autonomic dysfunctions, and other conditions. Methods Online literature searches were performed with Google, Google Scholar, PubMed, the bibliography from the Camp Detrick technical library, and at the Defense Technical Information Center. Reference lists in some of the primary research publications and reviews also provided source material. Search terms included botulinum, botulinus, and Camp Detrick. References related to the subsequent impacts of the Camp Detrick results were selected and cited from reviews and primary references in the more recent literature. Notes on toxin nomenclature and potential sources of error in this study are presented. Results The literature searches returned 27 citations of Camp Detrick authors, 24 of which were articles in peer-reviewed journals. The publications by these investigators included several disciplines such as biochemistry, immunology, pharmacology, physiology, and toxicology. A fundamental finding was the identification of critical nutritional components for improved growth of Clostridium botulinum and the increased production of BoTx serotype A. The purification processes that were developed at Camp Detrick allowed for the production of crystalline material to be scaled up for the manufacture of toxoid vaccine. Based on the research by Camp Detrick scientists, a toxoid supply of over 1 million units was available to vaccinate ~300,000 troops before the large-scale operations of D-Day. Conclusions BoTx research during the period 1943 to 1956 resulted in refinements in the techniques for isolating and purifying the crystalline BoTx type A. These results led to the development and manufacture of a toxoid vaccine that was available in a sufficient quantity to protect ~300,000 warfighters in a large-scale military operation. One of the most important long-term consequences derived from the knowledge gained by the efforts at Camp Detrick was the development in the 1980s of safe and effective therapeutic uses for BoTx type A, the most lethal biological substance known.

This paper combines an empirical analysis of national and international attempts to deal with the problem of poverty in China with a normative analysis of capability theory, as developed by Amartya Sen and Martha Nussbaum. In this way, it attempts to highlight the centrality of normative approaches to poverty and its elimination. The paper is in five parts. Section I analyses the history, development and effectiveness of social assistance under the Minimum Living Standards Scheme (MLSS) in China; Section II provides an outline of capability theory; Section III analyses the history, development and effectiveness of the United Nations Development Program's (UNDP) Millennium Development Goals, with a focus on the elimination of poverty in China; Section IV emphasises the importance of a capability approach to the measurement of poverty and outlines what needs to be done to reduce poverty in China; while Section V discusses what still needs to be done to promote an effective anti-poverty strategy in China.

Botulinum neurotoxins (BoNTs), the most poisonous proteins known to humankind, are a family of seven (serotype A to G) immunologically distinct proteins synthesized primarily by different strains of the anaerobic bacterium Clostridium botulinum. Being the causative agents of botulism, the toxins block neurotransmitter release by specifically cleaving one of the three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, thereby inducing flaccid paralysis. The development of countermeasures and therapeutics against BoNTs is a high-priority research area for public health because of their extreme toxicity and potential for use as biowarfare agents. Extensive research has focused on designing antagonists that block the catalytic activity of BoNTs. In this study, we screened 300 small natural compounds and their analogues extracted from Indian plants for their activity against BoNT serotype A (BoNT/A) as well as its light chain (LCA) using biochemical and cellular assays. One natural compound, a nitrophenyl psoralen (NPP), was identified to be a specific inhibitor of LCA with an in vitro 50% inhibitory concentration (IC50) value of 4.74 ± 0.03 µM. NPP was able to rescue endogenous synaptosome-associated protein 25 (SNAP-25) from cleavage by BoNT/A in human neuroblastoma cells with an IC50 of 12.2 ± 1.7 µM, as well as to prolong the time to the blocking of neutrally elicited twitch tensions in isolated mouse phrenic nerve-hemidiaphragm preparations. IMPORTANCE The long-lasting endopeptidase activity of BoNT is a critical biological activity inside the nerve cell, as it prompts proteolysis of the SNARE proteins, involved in the exocytosis of the neurotransmitter acetylcholine. Thus, the BoNT endopeptidase activity is an appropriate clinical target for designing new small-molecule antidotes against BoNT with the potential to reverse the paralysis syndrome of botulism. In principle, small-molecule inhibitors (SMIs) can gain entry into BoNT-intoxicated cells if they have a suitable octanol-water partition coefficient (log P) value and other favorable characteristics (P. Leeson, Nature 481:455–456, 2012, https://doi.org/10.1038/481455a). Several efforts have been made in the past to develop SMIs, but inhibitors effective under in vitro conditions have not in general been effective in vivo or in cellular models (L. M. Eubanks, M. S. Hixon, W. Jin, S. Hong, et al., Proc Natl Acad Sci U S A 104:2602–2607, 2007, https://doi.org/10.1073/pnas.0611213104). The difference between the in vitro and cellular efficacy presumably results from difficulties experienced by the compounds in crossing the cell membrane, in conjunction with poor bioavailability and high cytotoxicity. The screened nitrophenyl psoralen (NPP) effectively antagonized BoNT/A in both in vitro and ex vivo assays. Importantly, NPP inhibited the BoNT/A light chain but not other general zinc endopeptidases, such as thermolysin, suggesting high selectivity for its target. Small-molecule (nonpeptidic) inhibitors have better oral bioavailability, better stability, and better tissue and cell permeation than antitoxins or peptide inhibitors.

Persistently normal alanine aminotransferase (PNALT) is present in 30 to 40% of chronic hepatitis patients and it is generally accepted that they have no liver damage. However, some studies suggest that there are some degrees of mild to moderate histological liver damage. We evaluated the fibrosis stage and the virological outcome in treated patients with PNALT.

Botulism outbreak due to consumption of food contaminated with botulinum neurotoxins (BoNTs) is a public health emergency. The threat of bioterrorism through deliberate distribution in food sources and/or aerosolization of BoNTs raises global public health and security concerns due to the potential for high mortality and morbidity. Rapid and reliable detection methods are necessary to support clinical diagnosis and surveillance for identifying the source of contamination, performing epidemiological analysis of the outbreak, preventing and responding to botulism outbreaks. This review considers the applicability of various BoNT detection methods and examines their fitness-for-purpose in safeguarding the public health and security goals.

Aim: To describe a poorly known endoscopic entity associated with portal hypertension, characterized by polypoid lesions either in the stomach or small intestine of patients with cirrhosis. Methods: Between 2003 and 2012, patients with cirrhosis and portal hypertension underwent endoscopic workup of portal hypertension in our endoscopy unit. The clinical expression, endoscopic features of these lesions, and their pathological characteristics are described. Results: A total of 1538 patients were included, among which 14 (0.9%) presented polypoid lesions; these patients had evidenceofportalhypertensionandhaddilatedcapillariesinthelaminapropria.Fourpatientspresentedwithsevereanaemia or melaena and required treatment. Propranolol was administered to three patients, and one patient needed a transjugular intrahepatic portosystemic shunt in order to control bleeding. For asymptomatic patients in whom polypoid lesions were resected, no recurrence of lesions was observed during follow-up gastroscopy (median 36 months, range 7–85 months). Conclusion: Portal hypertension-associated gastric or small intestine polypoid lesions may be associated with a significant risk of bleeding and are responsive to adequate treatment of portal hypertension.

In this article, the authors present triggered instructions, a novel control paradigm for arrays of processing elements (PEs) aimed at exploiting spatial parallelism. Triggered instructions completely eliminate the program counter and allow programs to transition concisely between states without explicit branch instructions. They also allow efficient reactivity to inter-PE communication traffic. The approach provides a unified mechanism to avoid overserialized execution, essentially achieving the effect of techniques such as dynamic instruction reordering and multithreading, which each require distinct hardware mechanisms in a traditional sequential architecture.

Botulinum neurotoxin A (BoNT/A) is one of the most deadly toxins, and is the etiological agent of the potentially fatal condition, botulism. Herein, we investigated 8-hydroxyquinoline (quinolin-8-ol) as a potential inhibitor scaffold for preventing the deadly neurochemical effects of the toxin. Quinolinols are known chelators that can disrupt the BoNT/A metalloprotease zinc-containing active site, thus impeding its proteolysis of the endogenous protein substrate, synaptosomal-associated protein 25 (SNAP-25). Using this information, the structure-activity relationship (SAR) of the quinolinol-5-sulfonamide scaffold was explored through preparation of a crude sulfonamide library, and evaluating the library in a BoNT/A LC enzymatic assay. Potency optimization of the sulfonamide hit compounds was undertaken as informed by docking studies, granting a lead compound with a submicromolar Ki. These quinolinol analogues demonstrated inhibitory activity in a cell-based model for SNAP-25 cleavage and an ex vivo assay for BoNT/A-mediated muscle paralysis.

HCV core antigen (Ag) and HCV RNA levels were evaluated in matched liver biopsy samples and sera from 22 patients with hepatitis C infection by using the quantitative Architect HCV Ag immunoassay and a real-time RT-qPCR assay, respectively. The data showed a strong correlation between liver and serum compartments of HCV Ag levels ( r = 0.80) and HCV RNA levels ( r = 0.87). In summary, the serum HCV Ag and RNA levels reflect the intrahepatic values.

Background: Liver allocation in Eurotransplant (ET) is based on the MELD score. Interlaboratory MELD score differences in INR and creatinine determination have been reported. The clinical implication of this observation has not been demonstrated. Methods: MELD scores were calculated in 66 patients with liver cirrhosis using bilirubin, creatinine, and INR analyzed in six liver transplant centers. Based on allocation results of ET, patients transplanted from December 2006 to June 2007 were divided according to MELD score in four groups. For each group, the influence of the match MELD on the probability of receiving a transplant was studied (Cox proportional hazards model). Results: Laboratory-dependent significant differences in MELD score were demonstrated. Cox proportional hazards model showed a significant association between MELD score and the probability of organ allocation. The unadjusted hazard ratio for receiving a liver transplant was significantly different between group 2 and group 4 (group 2: MELD 19-24; group 4: MELD > 30). Conclusion: Laboratory-dependent significant differences in MELD score were observed between the six transplant centers. We demonstrated a significant association between the MELD score and the probability of organ allocation. The observed interlaboratory variation might yield a significant difference in organ allocation in patients with high MELD scores.

The botulinum neurotoxins (BoNT, serotypes A-G) are some of the most toxic proteins known and are the causative agents of botulism. Following exposure, the neurotoxin binds and enters peripheral cholinergic nerve endings and specifically and selectively cleaves one or more SNARE proteins to produce flaccid paralysis. This review centers on the kinetics of the Zn-dependent proteolytic activities of these neurotoxins, and briefly describes inhibitors, activators and factors underlying persistence of toxin action. Some of the structural, enzymatic and inhibitor data that are discussed here are available at the botulinum neurotoxin resource, BotDB (http://botdb.abcc.ncifcrf.gov).

This study was designed to test the hypothesis that the acute toxicity of the nerve agents S-[2-(diisopropylamino)ethyl]-O-ethyl methylphosphonothioate (VX), O-pinacolyl methylphosphonofluoridate (soman), and O-isopropyl methylphosphonofluoridate (sarin) in guinea pigs is age- and sex-dependent and cannot be fully accounted for by the irreversible inhibition of acetylcholinesterase (AChE). The subcutaneous doses of nerve agents needed to decrease 24-h survival of guinea pigs by 50% (LD50 values) were estimated by probit analysis. In all animal groups, the rank order of LD50 values was sarin > soman > VX. The LD50 value of soman was not influenced by sex or age of the animals. In contrast, the LD50 values of VX and sarin were lower in adult male than in age-matched female or younger guinea pigs. A colorimetric assay was used to determine the concentrations of nerve agents that inhibit in vitro 50% of AChE activity (IC50 values) in guinea pig brain extracts, plasma, red blood cells, and whole blood. A positive correlation between LD50 values and IC50 values for AChE inhibition would support the hypothesis that AChE inhibition is a major determinant of the acute toxicity of the nerve agents. However, such a positive correlation was found only between LD50 values and IC50 values for AChE inhibition in brain extracts from neonatal and prepubertal guinea pigs. These results demonstrate for the first time that the lethal potencies of some nerve agents in guinea pigs are age- and sex-dependent. They also support the contention that mechanisms other than AChE inhibition contribute to the lethality of nerve agents.

Experimental studies have demonstrated that botulinum neurotoxin serotype A (BoNT/A) causes flaccid paralysis by a multi-step mechanism. Following its binding to specific receptors at peripheral cholinergic nerve endings, BoNT/A is internalized by receptor-mediated endocytosis. Subsequently its zinc-dependent catalytic domain translocates into the neuroplasm where it cleaves a vesicle-docking protein, SNAP-25, to block neurally evoked cholinergic neurotransmission. We tested the hypothesis that mathematical models having a minimal number of reactions and reactants can simulate published data concerning the onset of paralysis of skeletal muscles induced by BoNT/A at the isolated rat neuromuscular junction (NMJ) and in other systems. Experimental data from several laboratories were simulated with two different models that were represented by sets of coupled, first-order differential equations. In this study, the 3-step sequential model developed by Simpson (J Pharmacol Exp Ther 212:16–21,1980) was used to estimate upper limits of the times during which anti-toxins and other impermeable inhibitors of BoNT/A can exert an effect. The experimentally determined binding reaction rate was verified to be consistent with published estimates for the rate constants for BoNT/A binding to and dissociating from its receptors. Because this 3-step model was not designed to reproduce temporal changes in paralysis with different toxin concentrations, a new BoNT/A species and rate (kS) were added at the beginning of the reaction sequence to create a 4-step scheme. This unbound initial species is transformed at a rate determined by kS to a free species that is capable of binding. By systematically adjusting the values of kS, the 4-step model simulated the rapid decline in NMJ function (kS ≥0.01), the less rapid onset of paralysis in mice following i.m. injections (kS = 0.001), and the slow onset of the therapeutic effects of BoNT/A (kS

Although hepatocellular carcinoma (HCC) has become a recognized indication for liver transplantation, the rules governing priority and access to the waiting list are not well defined. Patient- and tumor-related variables were evaluated in 226 patients listed primarily for HCC in Belgium, a region where the allocation system is patient-driven, priority being given to sicker patients, based on the Child-Turcotte-Pugh (CTP) score, Intention-to-treat and posttransplantation survival rates at 4 years were 56.5 and 66%, respectively, and overall HCC recurrence rate was 10%. The most significant predictors of failure to receive a transplant in due time were baseline CTP score equal to or above 9 (relative risk [RR] 4.1; confidence interval [Cl]: 1.7-9.9) and a fetoprotein above 100 ng/mL (RR 3.0; Cl: 1.2-7.1). Independent predictors of posttransplantation mortality were age equal to or above 50 years (RR 2.5; Cl: 1.0-3.7) and United Network for Organ Sharing pathological tumor nodule metastasis above the Milan criteria (RR 2.1; Cl: 1.0-5.9). Predictors of recurrence (10%) were a fetoprotein above 100 ng/mL (RR 3.2; Cl:1.1-10) and vascular involvement of the tumor on the explant (RR 3.6; Cl: 1.1-11.3). Assessing the value of the pretransplantation staging by imaging compared to explant pathology revealed 34% accuracy, absence of carcinoma in 8.3%, overstaging in 36.2%, and understaging in 10.4%. Allocation rules for HCC should consider not only tumor characteristics but also the degree of liver impairment. Patients older than 50 years with a stage above the Milan criteria at transplantation have a poorer prognosis after transplantation.

The seven antigenically distinct serotypes (A-G) of botulinum neurotoxin (BoNT) are responsible for the deadly disease botulism. BoNT serotype A (BoNT/A) exerts its lethal action by cleaving the SNARE protein SNAP-25, leading to inhibition of neurotransmitter release, flaccid paralysis and autonomic dysfunction. BoNTs are dichain proteins consisting of a ∼ 100 kDa heavy chain and a ∼ 50 kDa light chain; the former is responsible for neurospecific binding, internalization and translocation, and the latter for cleavage of neuronal SNARE proteins. Because of their extreme toxicity and history of weaponization, the BoNTs are regarded as potential biowarfare/bioterrorism agents. No post-symptomatic therapeutic interventions are available for BoNT intoxication other than intensive care; therefore it is imperative to develop specific antidotes against this neurotoxin. To this end, a cyclic peptide inhibitor (CPI-1) was evaluated in a FRET assay for its ability to inhibit BoNT/A light chain (Balc). CPI was found to be highly potent, exhibiting a Ki of 12.3 nM with full-length Balc448 and 39.2 nM using a truncated crystallizable form of the light chain (Balc424). Cocrystallization studies revealed that in the Balc424-CPI-1 complex, the inhibitor adopts a helical conformation, occupies a high percentage of the active site cavity and interacts in an amphipathic manner with critical active site residues. The data suggest that CPI-1 prevents SNAP-25 from accessing the Balc active site by blocking both the substrate binding path at the surface and the Zn(2+) binding region involved in catalysis. This differs from linear peptide inhibitors described to date which block only the latter.

Recombinant DNA technology and corresponding innovations in molecular biology, chemistry and medicine have led to novel therapeutic biomacromolecules as lead candidates in the pharmaceutical drug development pipelines. While monoclonal antibodies and other proteins provide therapeutic potential beyond the possibilities of small molecule drugs, the concomitant demand for supportive bioanalytical sample testing creates multiple novel challenges. For example, intact macromolecules can usually not be quantified by mass-spectrometry without enzymatic digestion and isotopically labeled internal standards are costly and/or difficult to prepare. Classical ELISA-type immunoassays, on the other hand, often lack the sensitivity required to obtain pharmacokinetics of low dosed drugs or pharmacodynamics of suitable biomarkers. Here we summarize emerging state-of-the-art ligand-binding assay technologies for pharmaceutical sample testing, which reveal enhanced analytical sensitivity over classical ELISA formats. We focus on immuno-PCR, which combines antibody specificity with the extremely sensitive detection of a tethered DNA marker by quantitative PCR, and alternative nucleic acid-based technologies as well as methods based on electrochemiluminescence or single-molecule counting. Using case studies, we discuss advantages and drawbacks of these methods for preclinical and clinical sample testing. © 2015 The Royal Society of Chemistry.

Botulinum neurotoxins (BoNTs) are extremely potent toxins that specifically cleave SNARE proteins in peripheral synapses, preventing neurotransmitter release. Neuronal responses to BoNT intoxication are traditionally studied by quantifying SNARE protein cleavage in vitro or monitoring physiological paralysis in vivo. Consequently, the dynamic effects of intoxication on synaptic behaviors are not well-understood. We have reported that mouse embryonic stem cell-derived neurons (ESNs) are highly sensitive to BoNT based on molecular readouts of intoxication. Here we study the time-dependent changes in synapse- and network-level behaviors following addition of BoNT/A to spontaneously active networks of glutamatergic and GABAergic ESNs. Whole-cell patch-clamp recordings indicated that BoNT/A rapidly blocked synaptic neurotransmission, confirming that ESNs replicate the functional pathophysiology responsible for clinical botulism. Quantitation of spontaneous neurotransmission in pharmacologically isolated synapses revealed accelerated silencing of GABAergic synapses compared to glutamatergic synapses, which was consistent with the selective accumulation of cleaved SNAP-25 at GAD1(+) pre-synaptic terminals at early timepoints. Different latencies of intoxication resulted in complex network responses to BoNT/A addition, involving rapid disinhibition of stochastic firing followed by network silencing. Synaptic activity was found to be highly sensitive to SNAP-25 cleavage, reflecting the functional consequences of the localized cleavage of the small subpopulation of SNAP-25 that is engaged in neurotransmitter release in the nerve terminal. Collectively these findings illustrate that use of synaptic function assays in networked neurons cultures offers a novel and highly sensitive approach for mechanistic studies of toxin:neuron interactions and synaptic responses to BoNT.

This article is a contribution to the occasional series dealing with a major book that influenced the author. Previous contributors include Stewart Macaulay, John Griffith, William Twining, Carol Harlow, Geoffrey Bindman, Harry Arthurs, and Andre-Jean Arnaud. It describes the author's original encounter and subsequent dialogue with Bureaucratic Justice, by Jerry L. Mashaw which transformed his thinking about administrative justice and shaped the research he conducted over the next 25 years. The article assesses the strengths and weaknesses of Mashaw's approach to administrative justice, describes the author's attempts to modify and extend Mashaw's approach, outlines four case studies conducted by the author, and describes how the United Kingdom government has recently adopted an approach to administrative justice that is very similar to Mashaw's. The article concludes by considering the implications of Mashaw's approach for researchers and policy makers who wish to promote the idea that governments should attempt to treat people fairly.

The article considers some unresolved policy choices associated with the implementation of ‘proportionate dispute resolution’, one of the most interesting ideas in the Department of Constitutional Affairs' White Paper Transforming Public Services: Complaints, Redress and Tribunals, published in 2004. It attempts to put the White Paper into context by tracing the Government's concern with tribunal reform over the last 50 years. It briefly compares the Franks Report, published in 1957, with the Leggatt Report, published in 2001, and outlines the steps that led to the publication, three years later, of the White Paper. It then analyses the similarities and differences in the approaches to reform taken by the Leggatt Report and the White Paper. The article focuses on the principle of ‘proportionate dispute resolution’, the idea that the ways in which cases are dealt with should reflect the nature of the dispute and what the person in dispute with a public body wishes to achieve. Seven policy options are considered, all of which hold out the prospect of enhancing administrative justice, either by reducing the incidence of disputes or by handling them more effectively. They are then assessed in terms of how well they are likely to do so.

The nerve agents soman, sarin, VX, and tabun are deadly organophosphorus (OP) compounds chemically related to OP insecticides. Most of their acute toxicity results from the irreversible inhibition of acetylcholinesterase (AChE), the enzyme that inactivates the neurotransmitter acetylcholine. The limitations of available therapies against OP poisoning are well recognized, and more effective antidotes are needed. Here, we demonstrate that galantamine, a reversible and centrally acting AChE inhibitor approved for treatment of mild to moderate Alzheimer’s disease, protects guinea pigs from the acute toxicity of lethal doses of the nerve agents soman and sarin, and of paraoxon, the active metabolite of the insecticide parathion. In combination with atropine, a single dose of galantamine administered before or soon after acute exposure to lethal doses of soman, sarin, or paraoxon effectively and safely counteracted their toxicity. Doses of galantamine needed to protect guinea pigs fully against the lethality of OPs were well tolerated. In preventing the lethality of nerve agents, galantamine was far more effective than pyridostigmine, a peripherally acting AChE inhibitor, and it was less toxic than huperzine, a centrally acting AChE inhibitor. Thus, a galantamine-based therapy emerges as an effective and safe countermeasure against OP poisoning.

We explore a model of time varying regional market integration that includes three factors for the North American equity market, the local Mexican equity market and the peso/dollar exchange rate. We argue that a useful instrument for the degree of integration is the sovereign yield spread. Applying our methodology to Mexico over the 1991–2002 period, we show that the degree of market integration was higher at the end of the period than at the beginning but that it exhibited wide swings that were related to both global as well as local events. We also discover that Mexico's currency risk is priced. Further, the currency returns process reveals strongly significant asymmetric volatility that is strongly related to the asymmetric volatility of the Mexican equity market returns process. A plausible reason for these results is that currency devaluations in emerging markets like Mexico can cause default-risk crises in local banking systems that mismatch local-currency assets and hard currency liabilities, whereas appreciations produce no such problems. Devaluations that destabilize banking systems are, therefore, more likely than appreciations to increase the volatilities of both the currency's and the equity market's returns.

Cannabinoid hyperemesis syndrome (CHS) is a rare constellation of clinical findings that includes a history of chronic heavy marijuana use, severe abdominal pain, unrelenting nausea, and intractable vomiting. A striking component of this history includes the use of hot showers or long baths that help to alleviate these symptoms. This is an underrecognized syndrome that can lead to expensive and unrevealing workups and can leave patients self-medicating their nausea and vomiting with the very substance that is causing their symptoms. Long-term treatment of CHS is abstinence from marijuana use—but the acute symptomatic treatment of CHS has been a struggle for many clinicians. Many standard medications used for the symptomatic treatment of CHS (including ondansetron, promethazine, and morphine) have repeatedly been shown to be ineffective. Here we present the use of lorazepam as an agent that successfully and safely treats the tenacious symptoms of CHS. Additionally, we build upon existing hypotheses for the pathogenesis of CHS to try to explain why a substance that has been used for thousands of years is only now beginning to cause this paradoxical hyperemesis syndrome.

A targeted delivery vehicle (DV) was developed for intracellular transport of emerging botulinum neurotoxin (BoNT) antagonists. The DV consisted of the isolated heavy chain (HC) of BoNT/A coupled to a 10-kDa amino dextran via the heterobifunctional linker 3-(2-pyridylthio)-propionyl hydrazide. The HC served to target BoNT-sensitive cells and promote internalization of the complex, while the dextran served as a platform to deliver model therapeutic molecules to the targeted cells. To determine the ability of this chimeric glycoprotein to enter neurons, dextran and HC were labeled independently with the fluorescent dyes Oregon green 488 and Cy3, respectively. Internalization of DV was monitored in primary cortical cells using laser confocal microscopy. Incubation of cells for 24 h with DV resulted in discrete punctate labeling of both soma and processes. The Cy3 and Oregon green 488 signals were generally co-localized, suggesting that the complex remained in the same intracellular compartment during the initial 24 h. The DV-associated fluorescence was reduced progressively by co-application of increasing concentrations of unlabeled BoNT/A holotoxin. The results suggest that the BoNT/A HC is able to mediate internalization of a coupled dextran, even though the latter bears no resemblance to the BoNT/A light chain (LC). The HC of BoNT/A thus offers promise as a selective carrier to deliver BoNT antagonists to the nerve terminal cytoplasm for inhibiting the proteolytic activity of internalized BoNT/A LC.

Primary dissociated fetal mouse spinal cord cultures were used to study the mechanisms underlying the differences in persistence of botulinum neurotoxin A (BoNT/A) and botulinum neurotoxin/E (BoNT/E) activities. Spinal cord cultures were exposed to BoNT/A (0.4 pM) for 2–3 days, which converted approximately half of the SNAP-25 to an altered form lacking the final nine C-terminal residues. The distribution of toxin-damaged to control SNAP-25 remained relatively unchanged for up to 80 days thereafter. Application of a high concentration of BoNT/E (250 pM) either 25 or 60 days following initial intoxication with BoNT/A converted both normal and BoNT/A-truncated SNAP-25 into a single population lacking the final 26 C-terminal residues. Excess BoNT/E was removed by washout, and recovery of intact SNAP-25 was monitored by Western blot analysis. The BoNT/E-truncated species gradually diminished during the ensuing 18 days, accompanied by the reappearance of both normal and BoNT/A-truncated SNAP-25. Return of BoNT/A-truncated SNAP-25 was observed in spite of the absence of BoNT/A in the culture medium during all but the first 3 days of exposure. These results indicate that proteolytic activity associated with the BoNT/A light chain persists inside cells for >11 weeks, while recovery from BoNT/E is complete in

Background & Aims: An absence or a presence of mutated transforming growth factor (TGF)-β receptors is a possible hypothesis explaining the resistance of cancer cells to the growth-inhibitory effect of TGF-β. Mutations involving microsatellite-like regions of the type II TGF-β receptor have been described in subgroups of colorectal cancers. The aim of this study was to investigate the expression and distribution of TGF-β receptors in sporadic colorectal cancers and normal tissues. Methods: Thirty-three sporadic colorectal cancers and 20 normal colonic tissues were explored by immunohistochemistry for the expression of type I and type II TGF-β receptors. Eighteen tumor and 20 normal samples were used for radioactive thermocycling and sequencing of the two microsatellite-like regions of the type II receptor. Results: Both receptors were overexpressed in tumors compared with normal samples. There was a relationship between the abundance of type II receptor expression and the degree of differentiation of the tumors but not the Dukes' staging or the localization of the neoplasias. No mutation was observed in the microsatellite-like regions of receptor II in any of the samples. Conclusions: Sporadic colorectal cancers do not show an absence or a presence of mutated TGF-β receptors that could explain a resistance to TGF-β–mediated growth inhibition. The pathways to tumorigenesis of sporadic colorectal cancers may be different from those of some hereditary ones. GASTROENTEROLOGY 1998;114:1211-1220

values are made explicit. In this paper, I look back on a programme of research on parental choice which I carried out more than 10 years ago and explain how this was related to the policy agenda at the time. After summarising the main findings, the research is subjected to some criticism-in particular for its parochialism and its failure to adopt a comparative perspective. The paper then reviews policy developments in Scotland and England since that time, describes the current policy agenda and indicates how further research might respond to this. It concludes that the key policy issue to which research should now be addressed is the relationship between choice and diversity and argues that this should be studied comparatively across a number of different educational systems through research which seeks to investigate the implications of each principle for the other and for other societal values.