Enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxins in foods.

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of staphylococcal enterotoxins in foods. The "double-antibody sandwich" protocol combines parts of several procedures reported previously. Horseradish peroxidase was conjugated to antibody specific for an enterotoxin, and the antibody-enzyme conjugate was assayed with a 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid)-H2O2 substrate solution. Enterotoxins were added to a variety of foods that were representative of those implicated in staphylococcal food poisoning outbreaks. Extracts of the foods were assayed by the ELISA and radioimmunoassay. Enterotoxin levels below 1 ng/g of food were consistently detectable by the ELISA. These results compared favorably with those of the radioimmunoassay. Experiments confirmed the interference of protein A in double-antibody sandwich ELISAs. Although protein A interference has not been demonstrated to be a problem in food extracts, we suggest a screen for protein A interference in which immunoglobulin G from nonimmunized rabbits is used. All of the known staphylococcal enterotoxins could be detected by this method. Analysis of a food product for entertoxin by the ELISA can be completed in an 8-h working day.     

Effect of fertilizer levels on grain yield,incidence and severity of downy mildew in pearl millet

The effect of various levels of nitrogen (0.0, 30.0, 60.0, 120.0) and phosphorus (0.0, 6.5, 13.0, 36.0) on the incidence and severity of downy mildew of pearl millet and yield of two pearl millet varieties (Zango and GB8375) were studied under field conditions in 2000 and 2001 respectively. Both nitrogen and phosphorus significantly increased incidence and severity of the disease in the two varieties. Grain yield and 1000 grain weight of the varieties also increased with nitrogen and phosphorus levels.     

The asymmetric function of Dph1–Dph2 heterodimer in diphthamide biosynthesis

JBIC Journal of Biological Inorganic Chemistry - Diphthamide, the target of diphtheria toxin, is a post-translationally modified histidine residue found in archaeal and eukaryotic translation...     

Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts

Transforming growth factor-β₁ (TGF-β₁) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β₁ may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β₁-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β₁ to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β₁ promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β₁ in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β₁-induced autophagy is required for the fibrogenic response in hATMyofbs.Interstitial fibrosis is common to many cardiovascular disease etiologies including myocardial infarction (MI),¹ diabetic cardiomyopathy² and hypertension.³ Fibrosis may arise due to maladaptive cardiac remodeling following injury and is a complex process resulting from activation of signaling pathways, such as TGF-β₁.⁴ TGF-β₁ signaling has broad-ranging effects that may affect cell growth, differentiation and the production of extracellular matrix (ECM) proteins.^{5, 6} Elevated TGF-β₁ is observed in post-MI rat heart⁷ and is associated with fibroblast-to-myofibroblast phenoconversion and concomitant activation of canonical Smad signaling.⁸ The result is a proliferation of myofibroblasts, which then leads to inappropriate deposition of fibrillar collagens, impaired cardiac function and, ultimately, heart failure.^{9, 10}Autophagy is necessary for cellular homeostasis and is involved in organelle and protein turnover.^{11, 12, 13, 14} Autophagy aids in cell survival by providing primary materials, for example, amino acids and fatty acids for anabolic pathways during starvation conditions.^{15, 16} Alternatively, autophagy may be associated with apoptosis through autodigestive cellular processes, cellular infection with pathogens or extracellular stimuli.^{17, 18, 19, 20} The overall control of cardiac fibrosis is likely due to the complex functioning of an array of regulatory factors, but to date, there is little evidence linking autophagy with fibrogenesis in cardiac tissue.^{11, 12, 13, 14, 15, 16, 17, 18, 21, 22}Recent studies have demonstrated that TGF-β₁ may not only promote autophagy in mouse fibroblasts and human tubular epithelial kidney cells^{15, 23, 24} but can also inhibit this process in fibroblasts extracted from human patients with idiopathic pulmonary fibrosis.²⁵ Moreover, it has recently been reported that autophagy can negatively¹⁵ and positively^{25, 26, 27} regulate the fibrotic process in different model cell systems. In this study, we have explored the putative link between autophagy and TGF-β₁-induced fibrogenesis in human atrial myofibroblasts (hATMyofbs) and in a model of MI rat heart.     

Strategies of Bringing Drug Product Marketing Applications to Meet Current Regulatory Standards

In the past decade, many guidance documents have been issued through collaboration of global organizations and regulatory authorities. Most of these are applicable to new products, but there is a risk that currently marketed products will not meet the new compliance standards during audits and inspections while companies continue to make changes through the product life cycle for continuous improvement or market demands. This discussion presents different strategies to bringing drug product marketing applications to meet current and emerging standards. It also discusses stability and method designs to meet process validation and global development efforts.At the 2014 American Association of Pharmaceutical Scientists (AAPS) annual meeting in San Diego, CA, Yan Wu (Merck) and Anita Freed (Pfizer) led a symposium entitled “Bringing Drug Product Marketing Applications to Current Regulatory Standards: Trials and Tribulations.” This symposium was very timely as this topic is a growing industry concern, evidenced by over 300 attendees, and considering the new guidances (1–8) that have been established over the past decade. While most of these quality standards are applicable to new drug products, there is a risk that currently marketed products, known as legacy products, will not meet the new compliance standards during audits and inspections. Companies also need to continuously make process or method changes for in-line products as part of product life cycle management efforts or to meet different market needs. If legacy (or in-line) products undergo a change, the question is how much extra effort is needed to have these products meet current standards to support the associated submission. This symposium addressed these issues and offered modeling tools using existing data or other approaches and case studies to effectively manage post-approval changes. Presentations included the following:

• Modeling historical data to support process and method stability changes
• Food and Drug Administration (FDA) perspectives on application of International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Q8 to legacy products
• Assessment of impact on stability with manufacturing, packaging, and/or method changes
• Applying Association of Southeast Asian Nations (ASEAN) stability requirements to legacy products and managing specifications across climatic zones

This paper provides an overview of the presentations and highlights strategies and points of consideration when bringing marketing applications of the legacy drug products to current and emerging standards.     

Modeling large regions in proteins: applications to loops, termini, and folding

Template-based methods for predicting protein structure provide models for a significant portion of the protein but often contain insertions or chain ends (InsEnds) of indeterminate conformation. The local structure prediction "problem" entails modeling the InsEnds onto the rest of the protein. A well-known limit involves predicting loops of ≤12 residues in crystal structures. However, InsEnds may contain as many as ~50 amino acids, and the template-based model of the protein itself may be imperfect. To address these challenges, we present a free modeling method for predicting the local structure of loops and large InsEnds in both crystal structures and template-based models. The approach uses single amino acid torsional angle "pivot" moves of the protein backbone with a C(β) level representation. Nevertheless, our accuracy for loops is comparable to existing methods. We also apply a more stringent test, the blind structure prediction and refinement categories of the CASP9 tournament, where we improve the quality of several homology based models by modeling InsEnds as long as 45 amino acids, sizes generally inaccessible to existing loop prediction methods. Our approach ranks as one of the best in the CASP9 refinement category that involves improving template-based models so that they can function as molecular replacement models to solve the phase problem for crystallographic structure determination.     

Plasma polyunsaturated fatty acids and regional cerebral glucose metabolism in major depression

Deficiencies in polyunsaturated essential fatty acids (PUFA) are implicated in mood disorders, although mechanisms of action and regional specificity in the brain are unknown. We hypothesized that plasma phospholipid PUFA levels are correlated with regionally specific relative cerebral metabolic rates of glucose (rCMRglu). Medication-free depressed subjects (N=29) were studied using [¹⁸F]-fluoro-2-deoxyglucose positron emission tomography. Docosahexaenoic acid (22:6n-3), arachidonic acid (20:4n-6), and eicosapentaenoic acid (20:5n-3) were assessed as a percentage of total phospholipid PUFA (DHA%, AA%, and EPA%, respectively). DHA% and AA% correlated positively with rCMRglu in temporoparietal cortex. In addition, DHA% correlated negatively with rCMRglu in prefrontal cortex and anterior cingulate. No correlations were seen with EPA%. Thus, under conditions of low plasma DHA, rCMRglu was higher in temporoparietal cortex and lower in anterior cingulate/prefrontal cortex. Opposing effects of DHA on these regions is a hypothesis that could be addressed in future prospective studies with n-3 supplementation. This pilot study is the first to demonstrate fatty acid and regionally specific correlations in the brain between plasma PUFA and rCMRglu in humans.     

A Vault Nanoparticle Vaccine Induces Protective Mucosal Immunity

Background

Generation of robust cell-mediated immune responses at mucosal surfaces while reducing overall inflammation is a primary goal for vaccination. Here we report the use of a recombinant nanoparticle as a vaccine delivery platform against mucosal infections requiring T cell-mediated immunity for eradication.

Methodology/Principal Findings

We encapsulated an immunogenic protein, the major outer membrane protein (MOMP) of Chlamydia muridarum, within hollow, vault nanocapsules (MOMP-vaults) that were engineered to bind IgG for enhanced immunity. Intranasal immunization (i.n) with MOMP-vaults induced anti-chlamydial immunity plus significantly attenuated bacterial burden following challenge infection. Vault immunization induced anti-chlamydial immune responses and inflammasome formation but did not activate toll-like receptors. Moreover, MOMP-vault immunization enhanced microbial eradication without the inflammation usually associated with adjuvants.

Conclusions/Significance

Vault nanoparticles containing immunogenic proteins delivered to the respiratory tract by the i.n. route can act as “smart adjuvants” for inducing protective immunity at distant mucosal surfaces while avoiding destructive inflammation.