The Structure of 5-Cyclohexyl-2′-Deoxyuridine as Studied by X-Ray Crystallography and NMR Spectroscopy

Abstract

5-Cyclohexyl-2′-deoxyuridine (I) is an example of a 5-substituted pyrimidine 2′-deoxynucleoside which exhibits no antiviral activity and which is not a substrate for either cellular or viral (herpes) kinases. Despite the fact that a cursory inspection of NMR spectra of the compound, taken in DMSO-d ₆ solution, suggested that the compound had a normal conformation, we here show that in the crystal and in aqueous solution (analysed by 2D NMR techniques), the conformation of this nucleoside has a syn-glycosidic and C4′-exo (₄E) sugar pucker conformation.     

Natural Infection of Burkholderia pseudomallei in an Imported Pigtail Macaque (Macaca nemestrina) and Management of the Exposed Colony

Identification of the select agent Burkholderia pseudomallei in macaques imported into the United States is rare. A purpose-bred, 4.5-y-old pigtail macaque (Macaca nemestrina) imported from Southeast Asia was received from a commercial vendor at our facility in March 2012. After the initial acclimation period of 5 to 7 d, physical examination of the macaque revealed a subcutaneous abscess that surrounded the right stifle joint. The wound was treated and resolved over 3 mo. In August 2012, 2 mo after the stifle joint wound resolved, the macaque exhibited neurologic clinical signs. Postmortem microbiologic analysis revealed that the macaque was infected with B. pseudomallei. This case report describes the clinical evaluation of a B. pseudomallei-infected macaque, management and care of the potentially exposed colony of animals, and protocols established for the animal care staff that worked with the infected macaque and potentially exposed colony. This article also provides relevant information on addressing matters related to regulatory issues and risk management of potentially exposed animals and animal care staff.Abbreviations: CDC, Centers for Disease Control and Prevention; IHA, indirect hemagglutination assay; PEP, postexposure prophylacticBurkholderia pseudomallei, formerly known as Pseudomonas pseudomallei, is a gram-negative, aerobic, bipolar, motile, rod-shaped bacterium. B. pseudomallei infections (melioidosis) can be severe and even fatal in both humans and animals. This environmental saprophyte is endemic to Southeast Asia and northern Australia, but it has also been found in other tropical and subtropical areas of the world.^7,22,32,42 The bacterium is usually found in soil and water in endemic areas and is transmitted to humans and animals primarily through percutaneous inoculation, ingestion, or inhalation of a contaminated source.^{8, 22,28,32,42} Human-to-human, animal-to-animal, and animal-to-human spread are rare.^8,32 In December 2012, the National Select Agent Registry designated B. pseudomallei as a Tier 1 overlap select agent.³⁹ Organisms classified as Tier 1 agents present the highest risk of deliberate misuse, with the most significant potential for mass casualties or devastating effects to the economy, critical infrastructure, or public confidence. Select agents with this status have the potential to pose a severe threat to human and animal health or safety or the ability to be used as a biologic weapon.³⁹Melioidosis in humans can be challenging to diagnose and treat because the organism can remain latent for years and is resistant to many antibiotics.^12,37,41 B. pseudomallei can survive in phagocytic cells, a phenomenon that may be associated with latent infections.^19,38 The incubation period in naturally infected animals ranges from 1 d to many years, but symptoms typically appear 2 to 4 wk after exposure.^13,17,35,38 Disease generally presents in 1 of 2 forms: localized infection or septicemia.²² Multiple methods are used to diagnose melioidosis, including immunofluorescence, serology, and PCR analysis, but isolation of the bacteria from blood, urine, sputum, throat swabs, abscesses, skin, or tissue lesions remains the ‘gold standard.’^9,22,40,42 The prognosis varies based on presentation, time to diagnosis, initiation of appropriate antimicrobial treatment, and underlying comorbidities.^7,28,42 Currently, there is no licensed vaccine to prevent melioidosis.There are several published reports of naturally occurring melioidosis in a variety of nonhuman primates (NHP; 2,10,13,17,25,30,31,35 The first reported case of melioidosis in monkeys was recorded in 1932, and the first published case in a macaque species was in 1966.³⁰ In the United States, there have only been 7 documented cases of NHP with B. pseudomallei infection.^2,13,17 All of these cases occurred prior to the classification of B. pseudomallei as a select agent. Clinical signs in NHP range from subclinical or subacute illness to acute septicemia, localized infection, and chronic infection. NHP with melioidosis can be asymptomatic or exhibit clinical signs such as anorexia, wasting, purulent drainage, subcutaneous abscesses, and other soft tissue lesions. Lymphadenitis, lameness, osteomyelitis, paralysis and other CNS signs have also been reported.^{2,7,10,22,28,32} In comparison, human''s clinical signs range from abscesses, skin ulceration, fever, headache, joint pain, and muscle tenderness to abdominal pain, anorexia, respiratory distress, seizures, and septicemia.^7,9,21,22

Table 1.

Summary of reported cases of naturally occurring Burkholderia pseudomalleiinfections in nonhuman primates

Amphibian skin fungal communities vary across host species and do not correlate with infection by a pathogenic fungus

Amphibian population declines caused by the fungus Batrachochytrium dendrobatidis (Bd) have prompted studies on the bacterial community that resides on amphibian skin. However, studies addressing the fungal portion of these symbiont communities have lagged behind. Using ITS1 amplicon sequencing, we examined the fungal portion of the skin microbiome of temperate and tropical amphibian species currently coexisting with Bd in nature. We assessed cooccurrence patterns between bacterial and fungal OTUs using a subset of samples for which bacterial 16S rRNA gene amplicon data were also available. We determined that fungal communities were dominated by members of the phyla Ascomycota and Basidiomycota, and also by Chytridiomycota in the most aquatic amphibian species. Alpha diversity of the fungal communities differed across host species, and fungal community structure differed across species and regions. However, we did not find a correlation between fungal diversity/community structure and Bd infection, though we did identify significant correlations between Bd and specific OTUs. Moreover, positive bacterial–fungal cooccurrences suggest that positive interactions between these organisms occur in the skin microbiome. Understanding the ecology of amphibian skin fungi, and their interactions with bacteria will complement our knowledge of the factors influencing community assembly and the overall function of these symbiont communities.     

Stoichiometry of the Sm proteins in yeast spliceosomal snRNPs supports the heptamer ring model of the core domain

Seven Sm proteins (B/B', D1, D2, D3, E, F and G proteins) containing a common sequence motif form a globular core domain within the U1, U2, U5 and U4/U6 spliceosomal snRNPs. Based on the crystal structure of two Sm protein dimers we have previously proposed a model of the snRNP core domain consisting of a ring of seven Sm proteins. This model postulates that there is only a single copy of each Sm protein in the core domain. In order to test this model we have determined the stoichiometry of the Sm proteins in yeast spliceosomal snRNPs. We have constructed seven different yeast strains each of which produces one of the Sm proteins tagged with a calmodulin-binding peptide (CBP). Further, each of these strains was transformed with one of seven different plasmids coding for one of the seven Sm proteins tagged with protein A. When one Sm protein is expressed as a CBP-tagged protein from the chromosome and a second protein was produced with a protein A-tag from the plasmid, the protein A-tag was detected strongly in the fraction bound to calmodulin beads, demonstrating that two different tagged Sm proteins can be assembled into functional snRNPs. In contrast when the CBP and protein A-tagged forms of the same Sm protein were co-expressed, no protein A-tag was detectable in the fraction bound to calmodulin. These results indicate that there is only a single copy of each Sm protein in the spliceosomal snRNP core domain and therefore strongly support the heptamer ring model of the spliceosomal snRNP core domain.     

Composition of symbiotic bacteria predicts survival in Panamanian golden frogs infected with a lethal fungus

Symbiotic microbes can dramatically impact host health and fitness, and recent research in a diversity of systems suggests that different symbiont community structures may result in distinct outcomes for the host. In amphibians, some symbiotic skin bacteria produce metabolites that inhibit the growth of Batrachochytrium dendrobatidis (Bd), a cutaneous fungal pathogen that has caused many amphibian population declines and extinctions. Treatment with beneficial bacteria (probiotics) prevents Bd infection in some amphibian species and creates optimism for conservation of species that are highly susceptible to chytridiomycosis, the disease caused by Bd. In a laboratory experiment, we used Bd-inhibitory bacteria from Bd-tolerant Panamanian amphibians in a probiotic development trial with Panamanian golden frogs, Atelopus zeteki, a species currently surviving only in captive assurance colonies. Approximately 30% of infected golden frogs survived Bd exposure by either clearing infection or maintaining low Bd loads, but this was not associated with probiotic treatment. Survival was instead related to initial composition of the skin bacterial community and metabolites present on the skin. These results suggest a strong link between the structure of these symbiotic microbial communities and amphibian host health in the face of Bd exposure and also suggest a new approach for developing amphibian probiotics.     

A dual function activity-dependent,muscle-specific enhancer from rat nicotinic acetylcholine receptor δ-subunit gene

Nicotinic acetylcholine receptors (nAChR) mediate communication between nerve and muscle. The expression of these receptors increases dramatically during muscle development when myoblasts are fusing into multinucleated myotubes. The molecular mechanisms mediating this muscle developmental stage specific expression are not well understood. We report here the identification of nAChR δ-subunit promoter DNA sequences that differentially interact with nuclear proteins isolated from myoblasts, myotubes, and nonmuscle cells. The functional role these sequences play in mediating muscle-specific expression was explored using mutagenesis and enhancer assays. These studies resulted in the identification of a 47-bp muscle-specific enhancer that mediates increased expression of the nAChR δ-submit gene during myotube formation. This enhancer contains an E-box and an element with similarity to the SV40 core enhancer (SVCE). Point mutations throughout this 47-bp enhancer showed that the E-box and the SVCE sequence are both necessary for conferring muscle-specific expression onto a heterologous promoter. Interestingly, this same DNA sequence also functions as an activity-dependent enhancer. © 1996 John Wiley & Sons, Inc.     

Social Immunity in Amphibians: Evidence for Vertical Transmission of Innate Defenses

Amphibian embryos are at risk of microbial infection. Here we find evidence that innate immune defenses, both antimicrobial skin peptides and mutualistic microbiota, of adult glass frogs, Hyalinobatrachium colymbiphyllum, can be transmitted to embryos deposited on leaves above rain forest streams in Panama and can inhibit the fungal pathogen Batrachochytrium dendrobatidis. Abstract in Spanish is available at http://www.blackwell‐synergy.com/loi/btp .     

97 Sensitivity of cyanobacteria to a potential biological control agent, bacterium SG-3

Cyanobacteria cause many problems in freshwater ecosystems. For example, the production of off-flavor compounds by cyanobacteria causes serious problems in catfish aquaculture. Control of cyanobacteria is generally limited to treatment with copper compounds, which are non-selective and sometimes ineffective at controlling certain species of cyanobacteria. Biological control could provide selective management by removing unwanted species while leaving desirable algae species. A bacterium (SG-3) (NRRL B-30043) lyses a number of planktonic species of cyanobacteria including bloom-forming species of Anabaena and Oscillatoria . We tested SG-3 for activity against 10 isolates, representing seven species, of mat-forming cyanobacteria within the genera Oscillatoria, Lyngbya , and Phormidium . Plugs (0.5 cm diameter) were cut from mats of the cyanobacterium, inoculated with liquid cultures of SG-3, and incubated as static cultures. The reduction in dry weights ranged from –0.5% to 90% compared to the untreated controls and appeared to be species specific. For example, dry weight reductions of Oscillatoria deflexoides and O. amoena ranged from 80 to 90% whereas the reduction of O. limosa tended to be lower at 36 to 72%. Although results varied among and within species, they indicate that this bacterium could have potential for use as a biological control for mat-forming cyanobacteria. Light microscopic observations indicate the bacteria do not penetrate the cyanobacteria cells. Currently, we are studying the possible causes of the observed cell lysis.     

A Call to Action for Mycetoma

Purpose of Review

Here, we discuss the current needs and priorities for mycetoma control and prevention, highlight lessons learned from leprosy and podoconiosis, and motivate an urgent need to accelerate progress toward reducing the burden of mycetoma in endemic areas.

Recent Findings

In 2015, the World Health Assembly (WHA) added mycetoma, a progressively debilitating disease caused by fungi and bacteria, to the World Health Organization (WHO) list of priority neglected tropical diseases (NTDs). Designation of other diseases as NTDs has raised awareness, enabled global partnerships, and advanced the capacity to combat disease through integrated programming. Although key mycetoma etiologic agents have been identified, many questions remain and mycetoma may similarly benefit from NTD designation.

Summary

In collaboration with experts at WHO and elsewhere, we formed a global mycetoma working group to connect partners from a variety of sectors and specialties. We envision that this group will evolve into a formalized partnership that can prioritize strategic planning, advocacy, and research needs, identify funding sources, and coordinate activities related to mycetoma and other NTDs affecting the skin. The experiences gained from other NTDs can help to guide the global mycetoma working group’s activities to better address the goals set forth in the WHA resolution.

     

Histidine availability is decisive in ROS-mediated cytotoxicity of copper complexes of Aβ1–16 peptide

The binding of metal ions to Aβ peptide plays an important role in the etiology of AD. Copper coordinates chiefly to His residues and produces reactive oxygen species (ROS) upon redox cycling. ROS builds enormous burden on the normal functioning of neuronal cells and results into deleterious effects. Recently, two structurally distinct copper binding sites with contrasting redox properties were characterized. Here, we demonstrate for the first time the effect of binding of two equivalents of Cu²⁺ on redox properties and cytotoxicity of Aβ peptide. Our electrochemical data and ascorbate consumption assay suggest that in the presence of two equivalents of copper; Aβ peptide has higher propensity of H₂O₂ generation. The oxidation of Aβ1–16 peptide due to both gamma radiolysis and metal catalyzed oxidation in the presence of two equivalents of copper is inhibited confirming the binding of both equivalents of copper to peptide. The electrochemical and cytotoxicity study shows that negative shift in the reduction potential is reflected as slightly higher cytotoxicity in SH-SY5Y cell lines for Aβ1–16–Cu²⁺ (1:2) complex.