Synthesis of lipopolysaccharide O side chains by Pseudomonas aeruginosa PAO1 requires the enzyme phosphomannomutase.

We have cloned a lipopolysaccharide (LPS) biosynthetic gene from Pseudomonas aeruginosa PAO1 that complements the defect in the production and incorporation of LPS O side chains in the LPS-rough strain AK1012. This gene was characterized by pulsed-field gel electrophoresis, deletion and restriction mapping of the cloned DNA, and biochemical analysis of the protein product. The cloned DNA was found to map to the 7-to-11-min region of the P. aeruginosa chromosome, and the gene needed for complementation of the LPS-rough phenotype was contained on a 2.6-kb HindIII-SacI fragment. This same size restriction fragment contains the alginate gene algC, which encodes the enzyme phosphomannomutase (PMM) and also maps to this region of the P. aeruginosa chromosome. The LPS-rough strain AK1012 was deficient in PMM activity, and this activity was restored to parental levels when the cloned gene was transferred to strain AK1012. In addition, the cloned gene could complement the PMM deficiency in the algC mutant strain 8858, and the cloned algC gene could restore the LPS-smooth phenotype to strain AK1012. These results indicate that the gene we have cloned is equivalent to the alginate gene algC. We designate this gene pmm to emphasize that it encodes the enzyme PMM, which has been shown to be essential for alginate production, and we demonstrate that PMM activity is required for the LPS-smooth phenotype in P. aeruginosa PAO1.     

A Radioimmunoassay-Based Method for Measuring the True Affinity of a Monoclonal Antibody with Trace Amounts of Radioactive Antigen: Illustration with the Products of a Cell-Free Protein Synthesis System

This communication describes a novel highly sensitive method for measuring the affinity of a monoclonal antibody for its antigen. It is based on a radioimmunoassay in which the antigen is labeled with radioactivity. It is therefore particularly well adapted to the study of trace amounts of radiolabeled polypeptide chains produced either in vivo, or in vitro by a cell free protein synthesis system or by chemical radiolabeling. It offers several advantages over previously described methods. Though making use of insolubilized antibody, it does measure the true affinity constant of the monoclonal antibody in solution for the antigen. It can be used even when the antigen is present at concentrations far below the dissociation constant of the antibody/antigen complex. It does not require the antigen or the antibody to be purified. In most cases, it requires no sophisticated equipment. This method could be easily adapted to the determination of the equilibrium constant of any type of protein/ligand system.     

Volatile sensation: The chemical ecology of the earthy odorant geosmin

Geosmin may be the most familiar volatile compound, as it lends the earthy smell to soil. The compound is a member of the largest family of natural products, the terpenoids. The broad distribution of geosmin among bacteria in both terrestrial and aquatic environments suggests that this compound has an important ecological function, for example, as a signal (attractant or repellent) or as a protective specialized metabolite against biotic and abiotic stresses. While geosmin is part of our everyday life, scientists still do not understand the exact biological function of this omnipresent natural product. This minireview summarizes the current general observations regarding geosmin in prokaryotes and introduces new insights into its biosynthesis and regulation, as well as its biological roles in terrestrial and aquatic environments.     

Framework for validation and implementation of in vitro toxicity tests

Summary The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technologic developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial, and regulatory communities, is recommended. Test validation acceptance is contingent on broad buy-in by disparate groups in the scientific community—academics, industry, and government. This is best achieved by early and frequent communication among parties and agreement on common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction, and refinement alternatives in toxicity testing.     

Vertically stratified frugivore community composition and interaction frequency in a liana fruiting across forest strata

Vertical stratification is a key feature of tropical forests and structures plant–frugivore interactions. However, it is unclear whether vertical differences in plant-frugivore interactions are due to differences among strata in plant community composition or inherent preferences of frugivores for specific strata. To test this, we observed fruit removal of a diverse frugivore community on the liana Marcgravia longifolia in a Peruvian rain forest. Unlike most other plants, Marcgravia longifolia produces fruits across forest strata. This enabled us to study effects of vertical stratification on fruit removal without confounding effects of plant species and stratum. We found a high number of visits of a few frugivore species in the understorey and a low number of visits of many different frugivores in the canopy and midstorey. Whereas partial and opportunistic frugivores foraged across strata with differing frequencies, obligate frugivores were only found eating fruits in the higher strata. Avian frugivores foraging in the canopy were mainly large species with pointed wings, whereas under- and midstorey avian foragers were smaller with rounded wings. Our findings suggest a continuous shift in the frugivore community composition along the vertical gradient, from a few generalized frugivores in the understorey to a diverse set of specialized frugivores in the canopy. This shift in the frugivore community leads to correlated, reciprocal changes from specialized to generalized plant-frugivore interactions. Thus, we conclude that vertical niche differentiation between species in tropical forests persists even when food resources are available across strata. This highlights its role for promoting biodiversity and ecosystem functioning.     

Treatment-resistant depression: definition,prevalence, detection,management, and investigational interventions

Treatment-resistant depression (TRD) is common and associated with multiple serious public health implications. A consensus definition of TRD with demonstrated predictive utility in terms of clinical decision-making and health outcomes does not currently exist. Instead, a plethora of definitions have been proposed, which vary significantly in their conceptual framework. The absence of a consensus definition hampers precise estimates of the prevalence of TRD, and also belies efforts to identify risk factors, prevention opportunities, and effective interventions. In addition, it results in heterogeneity in clinical practice decision-making, adversely affecting quality of care. The US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have adopted the most used definition of TRD (i.e., inadequate response to a minimum of two antidepressants despite adequacy of the treatment trial and adherence to treatment). It is currently estimated that at least 30% of persons with depression meet this definition. A significant percentage of persons with TRD are actually pseudo-resistant (e.g., due to inadequacy of treatment trials or non-adherence to treatment). Although multiple sociodemographic, clinical, treatment and contextual factors are known to negatively moderate response in persons with depression, very few factors are regarded as predictive of non-response across multiple modalities of treatment. Intravenous ketamine and intranasal esketamine (co-administered with an antidepressant) are established as efficacious in the management of TRD. Some second-generation antipsychotics (e.g., aripiprazole, brexpiprazole, cariprazine, quetiapine XR) are proven effective as adjunctive treatments to antidepressants in partial responders, but only the olanzapine-fluoxetine combination has been studied in FDA-defined TRD. Repetitive transcranial magnetic stimulation (TMS) is established as effective and FDA-approved for individuals with TRD, with accelerated theta-burst TMS also recently showing efficacy. Electroconvulsive therapy is regarded as an effective acute and maintenance intervention in TRD, with preliminary evidence suggesting non-inferiority to acute intravenous ketamine. Evidence for extending antidepressant trial, medication switching and combining antidepressants is mixed. Manual-based psychotherapies are not established as efficacious on their own in TRD, but offer significant symptomatic relief when added to conventional antidepressants. Digital therapeutics are under study and represent a potential future clinical vista in this population.     

Disruption of transport activity in a D93H mutant thiamine transporter 1, from a Rogers Syndrome family.

Rogers syndrome is an autosomal recessive disorder resulting in megaloblastic anemia, diabetes mellitus, and sensorineural deafness. The gene associated with this disease encodes for thiamine transporter 1 (THTR1), a member of the SLC19 solute carrier family including THTR2 and the reduced folate carrier (RFC). Using transient transfections into NIH3T3 cells of a D93H mutant THTR1derived from a Rogers syndrome family, we determined the expression, post-translational modification, plasma membrane targeting and thiamine transport activity. We also explored the impact on methotrexate (MTX) transport activity of a homologous missense D88H mutation in the human RFC, a close homologue of THTR1. Western blot analysis revealed that the D93H mutant THTR1 was normally expressed and underwent a complete N-glycosylation. However, while this mutant THTR1 was targeted to the plasma membrane, it was completely devoid of thiamine transport activity. Consistently, introduction into MTX transport null cells of a homologous D88H mutation in the hRFC did not result in restoration of MTX transport activity, thereby suggesting that D88 is an essential residue for MTX transport activity. These results suggest that the D93H mutation does not interfere with transporter expression, glycosylation and plasma membrane targeting. However, the substitution of this negatively charged amino acid (Asp93) by a positively charged residue (His) in an extremely conserved region (the border of transmembrane domain 2/intracellular loop 2) in the SLC19 family, presumably inflicts deleterious structural alterations that abolish thiamine binding and/or translocation. Hence, this functional characterization of the D93H mutation provides a molecular basis for Rogers syndrome.     

Zooplankton cohort analysis using systems identification techniques

The linear-transfer and lag-Manly models of zooplankton cohortdevelopment were examined using data generated from a thirdmore realistic model. The more realistic multi-transfer modelincluded variance in development rate among individuals. Thelinear-transfer model produced highly biased estimates of developmentrate under conditions of rapidly changing recruitment. Althoughits performance was improved by increasing the number of modeledstages and thus decreasing the rate of change in recruitmentcompared to stage duration, a positive bias remained. The lag-Manlymodel also produced positively biased estimates of stage durationgiven non-zero variance in development rates. A comparison ofthe models' performances under different simulated samplingregimes recommended the multi-transfer model. Use of the multi-transfermodel was illustrated by determining the development and mortalityrates of the brine shrimp, Artemia monica reared under threedifferent conditions of food and temperature corresponding tonatural regimes in Mono Lake, California. The experimental conditionsand sampling regime resulted in high relative standard errors(mean, 33%) in stage abundance estimates not atypical of zooplanktonsampling regimes in lakes. A Monte Carlo analysis was used todetermine the uncertainty in estimated parameters and determinethe level of stage aggregation which maximized the amount ofinformation derived from the experiments.     

Interaction of workplace demands and cardiovascular reactivity in progression of carotid atherosclerosis: population based study.

OBJECTIVE: To examine the combined influence of workplace demands and changes in blood pressure induced by stress on the progression of carotid atherosclerosis. DESIGN: Population based follow up study of unestablished as well as traditional risk factors for carotid atherosclerosis, ischaemic heart disease, and other outcomes. SETTING: Eastern Finland. SUBJECTS: 591 men aged 42-60 who were fully employed at baseline and had complete data on the measures of carotid atherosclerosis, job demands, blood pressure reactivity, and covariates. MAIN OUTCOME MEASURES: Change in ultrasonographically assessed intima-media thickness of the right and left common carotid arteries from baseline to 4 year follow up. RESULTS: Significant interactions between workplace demands and stress induced reactivity were observed for all measures of progression (P < 0.04). Men with large changes in systolic blood pressure (20 mm Hg or greater) in anticipation of a maximal exercise test and with high job demands had 10-40% greater progression of mean (0.138 v 0.123 mm) and maximum (0.320 v 0.261 mm) intima-media thickness and plaque height (0.347 v 0.264) than men who were less reactive and had fewer job demands. Similar results were obtained after excluding men with prevalent ischaemic heart disease at baseline. Findings were strongest among men with at least 20% stenosis or non-stenotic plaque at baseline. In this subgroup reactive men with high job demands had more than 46% greater atherosclerotic progression than the others. Adjustment for atherosclerotic risk factors did not alter the results. CONCLUSIONS: Men who showed stress induced blood pressure reactivity and who reported high job demands experienced the greatest atherosclerotic progression, showing the association between dispositional risk characteristics and contextual determinants of disease and suggesting that behaviourally evoked cardiovascular reactivity may have a role in atherogenesis.