Synthesis of cyclooctapeptides: constraints analogues of the peptidic neurotoxin, ω‐agatoxine IVB—an experimental point of view

ω‐AGA IVB is an important lead structure when considering the design of effectors of glutamate release inducting P/Q‐type calcium channels. The best route to achieve the analogues possessing the three‐dimensional arrangement corresponding to the native binding loop was the introduction of constraint by ring formation via side chain to side chain lactamization for suitably protected Lys and Glu residues. Since tryptophane residue located at position 14 of this neuropeptide has been suggested as essential for binding, analogues in which this amino acid was replaced by aza‐tryptophane and alanine were synthesized. The synthesis was carried out on various acid‐labile resins (BARLOS chlorotrityl, Rink amide, PEG‐based or Wang resins), by Fmoc strategy. In this paper, we describe optimization of the peptide cyclization with various protecting groups, and on resin or in solution cyclization experimental parameters. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.     

Defining the importance of phosphatidylserine synthase-1 (PSS1): unexpected viability of PSS1-deficient mice

Phosphatidylserine (PS) is a quantitatively minor, but physiologically important, phospholipid in mammalian cells. PS is synthesized by two distinct base-exchange enzymes, PS synthase-1 (PSS1) and PS synthase-2 (PSS2), that are encoded by different genes. PSS1 exchanges serine for choline of phosphatidylcholine, whereas PSS2 exchanges ethanolamine of phosphatidylethanolamine for serine. We previously generated mice lacking PSS2 (Bergo, M. O., Gavino, B. J., Steenbergen, R., Sturbois, B., Parlow, A. F., Sanan, D. A., Skarnes, W. C., Vance, J. E., and Young, S. G. (2002) J. Biol. Chem. 277, 47701-47708) and found that PSS2 is not required for mouse viability. We have now generated PSS1-deficient mice. In light of the markedly impaired survival of Chinese hamster ovary cells lacking PSS1 we were surprised that PSS1-deficient mice were viable, fertile, and had a normal life span. Total serine-exchange activity (contributed by PSS1 and PSS2) in tissues of Pss1(-/-) mice was reduced by up to 85%, but except in liver, the PS content was unaltered. Despite the presumed importance of PS in the nervous system, the rate of axonal extension of PSS1-deficient neurons was normal. Intercrosses of Pss1(-/-) mice and Pss2(-/-) mice yielded mice with three disrupted Pss alleles but no double knockout mice. In Pss1(-/-)/Pss2(-/-) and Pss1(-/-)/Pss2(-/-) mice, serine-exchange activity was reduced by 65-91%, and the tissue content of PS and phosphatidylethanolamine was also decreased. We conclude that (i) elimination of either PSS1 or PSS2, but not both, is compatible with mouse viability, (ii) mice can tolerate as little as 10% of normal total serine-exchange activity, and (iii) mice survive with significantly reduced PS and phosphatidylethanolamine content.     

Methylphenidate decreased the amount of glucose needed by the brain to perform a cognitive task

The use of stimulants (methylphenidate and amphetamine) as cognitive enhancers by the general public is increasing and is controversial. It is still unclear how they work or why they improve performance in some individuals but impair it in others. To test the hypothesis that stimulants enhance signal to noise ratio of neuronal activity and thereby reduce cerebral activity by increasing efficiency, we measured the effects of methylphenidate on brain glucose utilization in healthy adults. We measured brain glucose metabolism (using Positron Emission Tomography and 2-deoxy-2[18F]fluoro-D-glucose) in 23 healthy adults who were tested at baseline and while performing an accuracy-controlled cognitive task (numerical calculations) given with and without methylphenidate (20 mg, oral). Sixteen subjects underwent a fourth scan with methylphenidate but without cognitive stimulation. Compared to placebo methylphenidate significantly reduced the amount of glucose utilized by the brain when performing the cognitive task but methylphenidate did not affect brain metabolism when given without cognitive stimulation. Whole brain metabolism when the cognitive task was given with placebo increased 21% whereas with methylphenidate it increased 11% (50% less). This reflected both a decrease in magnitude of activation and in the regions activated by the task. Methylphenidate's reduction of the metabolic increases in regions from the default network (implicated in mind-wandering) was associated with improvement in performance only in subjects who activated these regions when the cognitive task was given with placebo. These results corroborate prior findings that stimulant medications reduced the magnitude of regional activation to a task and in addition document a "focusing" of the activation. This effect may be beneficial when neuronal resources are diverted (i.e., mind-wandering) or impaired (i.e., attention deficit hyperactivity disorder), but it could be detrimental when brain activity is already optimally focused. This would explain why methylphenidate has beneficial effects in some individuals and contexts and detrimental effects in others.     

A C-terminal aldehyde insect kinin analog enhances inhibition of weight gain and induces significant mortality in Helicoverpa zea larvae

The first reported examples of C-terminal aldehyde analogs of an insect neuropeptide are described. They are hexapeptide insect kinin analogs Boc-VFFPWG-H and Fmoc-RFFPWG-H. Activity observed for these modified analogs in an in vitro insect diuretic assay confirms that the C-terminal aldehyde group is tolerated by an insect kinin receptor. The two analogs demonstrate greatly enhanced activity over standard C-terminal amide insect kinins in a larval weight gain inhibition assay in the corn earworm Helicoverpa zea. Treatment with Boc-VFFPWG-H led to significant increases in larval mortality at doses of 500pm (45%) and 5nm (67%). Boc-VFFPWG-H represents a lead analog in the development of novel, environmentally friendly pest insect management agents based on the insect kinin class of neuropeptides.     

The new higher level classification of eukaryotes with emphasis on the taxonomy of protists

This revision of the classification of unicellular eukaryotes updates that of Levine et al. (1980) for the protozoa and expands it to include other protists. Whereas the previous revision was primarily to incorporate the results of ultrastructural studies, this revision incorporates results from both ultrastructural research since 1980 and molecular phylogenetic studies. We propose a scheme that is based on nameless ranked systematics. The vocabulary of the taxonomy is updated, particularly to clarify the naming of groups that have been repositioned. We recognize six clusters of eukaryotes that may represent the basic groupings similar to traditional "kingdoms." The multicellular lineages emerged from within monophyletic protist lineages: animals and fungi from Opisthokonta, plants from Archaeplastida, and brown algae from Stramenopiles.     

Assessing the effectiveness of a community intervention for monkeypox prevention in the Congo basin

Background

In areas where health resources are limited, community participation in the recognition and reporting of disease hazards is critical for the identification of outbreaks. This is particularly true for zoonotic diseases such as monkeypox that principally affect people living in remote areas with few health services. Here we report the findings of an evaluation measuring the effectiveness of a film-based community outreach program designed to improve the understanding of monkeypox symptoms, transmission and prevention, by residents of the Republic of the Congo (ROC) who are at risk for disease acquisition.

Methodology/Principal Findings

During 90 days, monkeypox outreach was conducted for ∼23,860 people in northern ROC. Two hundred seventy-one attendees (selected via a structured sample) were interviewed before and after participating in a small-group outreach session. The proportion of interviewees demonstrating monkeypox-specific knowledge before and after was compared. Significant gains were measured in areas of disease recognition, transmission, and mitigation of risk. The ability to recognize at least one disease symptom and a willingness to take a family member with monkeypox to the hospital increased from 49 and 45% to 95 and 87%, respectively (p<0.001, both). Willingness to deter behaviors associated with zoonotic risk, such as eating the carcass of a primate found dead in the forest, remained fundamentally unchanged however, suggesting additional messaging may be needed.

Conclusions/Significance

These results suggest that our current program of film-based educational activities is effective in improving disease-specific knowledge and may encourage individuals to seek out the advice of health workers when monkeypox is suspected.     

Improving Quality Control of Contagious Caprine Pleuropneumonia Vaccine with Tandem Mass Spectrometry

Vaccines to protect livestock against contagious caprine pleuropneumonia (CCPP) consist of inactivated, adjuvanted antigens. Quality control of these vaccines is challenging as total protein quantification provides no indication of protein identity or purity, and culture is not an option. Here, a tandem mass spectrometry approach is used to identify the mycoplasma antigen contained in reference samples and in commercial CCPP vaccines. By the same approach, the relative amounts of mycoplasma antigen and residual proteins originating from the production medium are determined. Mass spectrometry allows easy and rapid identification of the peptides present in the vaccine samples. Alongside the most probable mycoplasma species effectively present in the vaccines, a very high proportion of peptides from medium constituents are detected in the commercial vaccines tested.     

Cytoskeletal dynamics underlying collateral membrane protrusions induced by neurotrophins in cultured Xenopus embryonic neurons

The establishment and refinement of neuronal connections depend on dynamic modification of the morphology and physiology of developing axons in response to extrinsic factors. In embryonic cultures of Xenopus spinal neurons, acute application of brain-derived neurotrophic factor (BDNF) induced rapid collateral protrusion of filopodium-like microspikes and lamellipodia along the neurite processes, leading to a morphologic alternation of the neuron. Both types of membrane protrusions contained high concentrations of actin filaments and depended on the polymerization of the actin cytoskeleton. Immunofluorescent staining, however, revealed the presence of microtubules (MTs) in lamellipodia induced by BDNF. These MTs appeared to have arisen from debundling of MTs in the neurite shaft at the protrusion sites, splaying and extending in the rapidly protruding lamellipodia. Inhibition of microtubule polymerization by nocodazole largely abolished the formation of lamellipodia but not of microspikes. Taken together, our results suggest that collateral sprouting of microspikes and lamellipodia involve distinctly different cytoskeletal mechanisms. Although the actin cytoskeleton is solely responsible for microspike formation, cooperative efforts by microtubules and actin filaments are essential for lamellipodial protrusion in response to extrinsic factors.     

Controlling Neglected Tropical Diseases (NTDs) in Haiti: Implementation Strategies and Evidence of Their Success

Lymphatic filariasis (LF) and soil-transmitted helminths (STH) have been targeted since 2000 in Haiti, with a strong mass drug administration (MDA) program led by the Ministry of Public Health and Population and its collaborating international partners. By 2012, Haiti’s neglected tropical disease (NTD) program had reached full national scale, and with such consistently good epidemiological coverage that it is now able to stop treatment for LF throughout almost all of the country. Essential to this success have been in the detail of how MDAs were implemented. These key programmatic elements included ensuring strong community awareness through an evidence-based, multi-channel communication and education campaign facilitated by voluntary drug distributors; strengthening community trust of the drug distributors by ensuring that respected community members were recruited and received appropriate training, supervision, identification, and motivation; enforcing a “directly observed treatment” strategy; providing easy access to treatment though numerous distribution posts and a strong drug supply chain; and ensuring quality data collection that was used to guide and inform MDA strategies. The evidence that these strategies were effective lies in both the high treatment coverage obtained– 100% geographical coverage reached in 2012, with almost all districts consistently achieving well above the epidemiological coverage targets of 65% for LF and 75% for STH—and the significant reduction in burden of infection– 45 communes having reached the target threshold for stopping treatment for LF. By taking advantage of sustained international financial and technical support, especially during the past eight years, Haiti’s very successful MDA campaign resulted in steady progress toward LF elimination and development of a strong foundation for ongoing STH control. These efforts, as described, have not only helped establish the global portfolio of “best practices” for NTD control but also are poised to help solve two of the most important future NTD challenges—how to maintain control of STH infections after the community-based LF “treatment platform” ceases and how to ensure appropriate morbidity management for patients currently suffering from lymphatic filarial disease.