Engineering the activity and lifetime of heterogeneous catalysts for carbon nanotube growth via substrate ion beam bombardment.

We demonstrate that argon ion bombardment of single crystal sapphire leads to the creation of substrates that support the growth of vertically aligned carbon nanotubes from iron catalysts with a density, height, and quality equivalent to those grown on conventional, disordered alumina supports. We quantify the evolution of the catalyst using a range of surface characterization techniques and demonstrate the ability to engineer and pattern the catalyst support through control of ion beam bombardment parameters.

Experimental exposure of adult San Marcos salamanders and larval leopard frogs to the cercariae of Centrocestus formosanus.

The gill parasite Centrocestus formosanus (Trematoda: Heterophyidae) is an exotic parasite of concern in Texas because it has been shown to infect multiple threatened and endangered fish species. The purpose of this study was to determine if C. formosanus could present a threat to larval anurans, as well as threatened neotenic salamanders endemic to the spring-fed systems of Texas. We exposed adults of the San Marcos salamander Eurycea nana (Caudata: Plethodontidae) and tadpoles of the Rio Grande leopard frog Lithobates berlandieri (Anura: Ranidae) to the cercariae of C. formosanus . The San Marcos salamander showed no signs of metacercarial infection, suggesting that E. nana may be refractory to C. formosanus cercariae. Centrocestus formosanus readily infects the gills of leopard frog tadpoles, but the metacercariae apparently died prior to reaching maturity in our tadpoles.

Specialty compounding for improved patient care: 2006 national survey of compounding pharmacists.

Pharmacy compounding is re-emerging as a critical area of pharmacy practice. This re-emergence has generated considerable discussion among stakeholders of the pharmacy profession about how pharmacy compounding is best regulated to ensure the safety and efficacy of compounded preparations. In light of this discussion, the American College of Apothecaries conducted a survey in August 2006 that was sent to compounding pharmacists, the list of which was provided by the International Journal of Pharmaceutical Compounding. The purpose of the survey was to (1)assess the safety and efficacy of compounded preparations as reported by compounding pharmacists, (2)describe the types of compounding and other professional services that are offered by these pharmacists, (3)describe selected business information for compounding pharmacies, and (4)monitor changes in the compounding pharmacy landscape since the 2005 National Survey of Compounding Pharmacists, the results of which were published in the November/December 2006 issue of the International Journal of Pharmaceutical Compounding. State boards of pharmacy were surveyed for reports of problems related to instability of compounded preparations and adverse reactions caused by compounded preparations. Data trends in this study are similar to those reported in the 2005 survey, also conducted by the American College of Apothecaries. Repsondents reported relatively few adverse events caused by compounded preparations. These respondents, many of whom are independent pharmacists, offer a variety of compounding, professional, and patient care services. Of 18 responding state boards of pharmacy, four noted reports of compounding problems in their state.

A nucleic acid sequence-based amplification assay for real-time detection of norovirus genogroup II.

AIMS: To use molecular beacon based nucleic acid sequence-based amplification (NASBA) to develop a rapid, sensitive, specific detection method for norovirus (NV) genogroupII (GII). METHODS AND RESULTS: A method to detect NV GII from environmental samples using real-time NASBA was developed. This method was routinely sensitive to 100 copies of target RNA and intermittent amplification occurred with as few as 10 copies. Quantitative estimates of viral load were possible over at least four orders of magnitude. CONCLUSIONS: The NASBA method described here is a reliable and sensitive assay for the detection of NV. This method has the potential to be linked to a handheld NASBA device that would make this real-time assay a portable and inexpensive alternative to bench-top, lab-based assays. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of the real-time NASBA assay described here has resulted in a simple, rapid (<1 h), convenient testing format for NV. To our knowledge, this is the first example of a molecular beacon based NASBA assay for NV.

Specialty compounding for improved patient care: a national survey of compounding pharmacists.

The apparent re-emergence of pharmaceutical compounding as an important part of health care is largely explained by the increasing demand for pharmaceutical preparations that are not commercially available. However, there have been recent discussions among regulatory agencies and various stakeholders of the pharmacy profession as to the safety and efficacy of pharmaceutical compounding, and how compounding should best be regulated. In light of this discussion, the American College of Apothecaries conducted a survey of International Journal of Pharmaceutical Compounding subscribers in July 2005 to (1) assess the safety and efficacy of compounded preparations, (2) describe the types of compounding and other professional services that are offered by pharmacists, and (3) describe selected compounding pharmacies. In general, respondents reported few incidents of adverse events resulting from compounded preparations, and reported having offered a variety of compounding, professional, and patient care specialties services.

Egg shell ultrastructure of the fish nematode Huffmanela huffmani (Trichosomoididae).

The egg shell of Huffmanela huffmani Moravec, 1987 forms three main layers: an outer vitelline layer, a middle chitinous layer, and an inner lipid layer. The vitelline layer, forming the superficial projections of the egg shell, comprises two parts: an outer electron-dense, and an inner electron-lucid part. The chitinous layer is differentiated into three parts: an outer homogenous electron-dense part, a lamellated part, and an inner electron-dense net-like part. The lipid layer comprises an outer net-like electron-lucid part, and an inner homogenous electron-lucid part. The polar plugs are formed by electron-lucid material with fine electron-dense fibrils.

Characterization of the binding interface between the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase.

The interaction of the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase, Ccc2a, was investigated by NMR in solution. In particular, a solution of Cu(I)-15NAtx1 was titrated with apo-Ccc2a, and, vice versa, a solution of Cu(I)-15NCcc2a was titrated with apo-Atx1. By following the 15N and 1H chemical shifts, a new species is detected in both experiments. This species is the same in both titrations and is in fast exchange with the parent species on the NMR time scale. Nuclear relaxation data are consistent with the formation of an adduct. Judging from the nuclear Overhauser effect spectroscopy patterns, the structure of Cu(I)-15NCcc2a in the presence of apo-Atx1 is not significantly altered, whereas Cu(I)-15NAtx1 in the presence of apo-Ccc2a experiences some changes with respect to both the apoproteins and the Cu(I)-loaded proteins. The structure of the Cu(I)-15NAtx1 moiety in the adduct was obtained from 1137 nuclear Overhauser effects to a final root mean square deviation to the mean structure of 0.76 +/- 0.13 A for the backbone and 1.11 +/- 0.11 A for the heavy atoms. 15N and 1H chemical shifts suggest the regions of interaction that, together with independent information, allow a structural model of the adduct to be proposed. The apo form of Atx1 displays significant mobility in loops 1 and 5, the N-terminal part of helix alpha1, and the C-terminal part of helix alpha2 on the ms-micros time scale. These regions correspond to the metal binding site. Such mobility is largely reduced in the free Cu(I)-Atx1 and in the adduct with apo-Ccc2a. The analogous mobility of Ccc2a in both Cu(I) and apo forms is reduced with respect to Atx1. Such an adduct is relevant as a structural and kinetic model for copper transfer from Atx1 to Ccc2a in physiological conditions.

Observations on the biology of Rhabdochona kidderi texensis, a parasite of North American cichlids.

An examination of a sample of benthic invertebrates collected from the Upper San Marcos River in southwestern Texas, USA in September 1999 revealed that the nymph of the ephemeropteran Tricorythodes curvatus served as natural intermediate host of the nematode Rhabdochona kidderi texensis (Nematoda: Rhabdochonidae), an intestinal parasite mainly of the Rio Grande perch (Cichlasoma cyanoguttatum) in this locality; the prevalence of the parasite's third- and fourth-stage larvae in mayflies was 6.8% with the intensity of 1-2 larvae per nymph. Live R. kidderi texensis eggs collected from nematodes recovered from C. cyanoguttatum in Texas were transported to the Czech Republic, where they were used to experimentally infect nymphs of the palaearctic mayfly species Paraleptophlebia submarginata; the development of infective third- and fourth-stage larvae in this experimental intermediate host was completed after approximately 10 days at 19 degrees C. Infected nymphs were fed to aquarium-reared fishes, four Cichlasoma nigrofasciatum and one Oreochromis niloticus, of which only three of the former became infected. The last (fourth) moult of a male nematode was observed in C. nigrofasciatum 23 days p.i. and adult males and gravid females with not fully mature (non-embryonated) eggs in uteri on days 40 and 51 p.i. The prepatent period of R. kidderi texensis is approximately two months.

Function, structure, and mechanism of intracellular copper trafficking proteins.

Genetic, biochemical, and spectroscopic studies have established a new function for an intracellular protein, i.e., guiding and inserting a copper cofactor into the active site of a target enzyme. Studies of these new proteins have revealed a fundamental aspect of copper physiology, namely the vast overcapacity of the cytoplasm for copper sequestration. This finding framed the mechanistic, energetic, and structural aspects of intracellular copper trafficking proteins. One hallmark of the copper chaperones is the similarity of the protein fold between the chaperone and its target enzyme. The surface residues presented by each partner, however, are quite different, and some initial findings concerning the complementarity of these interfaces have led to mechanistic insights. The copper chaperones appear to lower the activation barrier for metal transfer into specific protein-binding sites. The manner in which they facilitate metal insertion appears to involve a docking of the metal donor and acceptor sites in close proximity to one another. Although the intimate mechanism is still open, it appears that a low activation barrier for metal transfer is achieved by a network of coordinate-covalent, electrostatic, and hydrogen bonding interactions in the vicinity of the metal-binding site itself.

The independent cue and cus systems confer copper tolerance during aerobic and anaerobic growth in Escherichia coli.

Copper is essential but can be toxic even at low concentrations. Coping with this duality requires multiple pathways to control intracellular copper availability. Three copper-inducible promoters, controlling expression of six copper tolerance genes, were recently identified in Escherichia coli. The cue system employs an inner membrane copper transporter, whereas the cus system includes a tripartite transporter spanning the entire cell envelope. Although cus is not essential for aerobic copper tolerance, we show here that a copper-sensitive phenotype can be observed when cus is inactivated in a cueR background. Furthermore, a clear copper-sensitive phenotype for the cus system is revealed in the absence of O(2). These results indicate that the cue pathway, which includes a copper exporter, CopA, and a periplasmic oxidase, CueO, is the primary aerobic system for copper tolerance. During anaerobic growth, however, copper toxicity increases, and the independent cus copper exporter is also necessary for full copper tolerance. We conclude that the cytosolic (CueR) and periplasmic (CusRS) sensor systems differentially regulate copper export systems in response to changes in copper and oxygen availability. These results underscore the increased toxicity of copper under anaerobic conditions and the complex adaptation of copper export in E. coli.

Solution structure of the Cu(I) and apo forms of the yeast metallochaperone, Atx1.

The (1)H NMR solution structure of the Cu(I)-bound form of Atx1, a 73-amino acid metallochaperone protein from the yeast Saccharomyces cerevisiae, has been determined. Ninety percent of the (1)H and 95% of the (15)N resonances were assigned, and 1184 meaningful NOEs and 42 (3)J(HNH)(alpha) and 60 (1)J(HN) residual dipolar couplings provided a family of structures with rmsd values to the mean structure of 0.37 +/- 0.07 A for the backbone and 0.83 +/- 0.08 A for all heavy atoms. The structure is constituted by four antiparallel beta strands and two alpha helices in a betaalphabetabetaalphabeta fold. Following EXAFS data [Pufahl, R., Singer, C. P., Peariso, K. L., Lin, S.-J., Schmidt, P. J., Fahrni, C. J., Cizewski Culotta, V., Penner-Hahn, J. E., and O'Halloran, T. V. (1997) Science 278, 853-856], a copper ion can be placed between two sulfur atoms of Cys15 and Cys18. The structure of the reduced apo form has also been determined with similar resolution using 1252 meaningful NOEs (rmsd values for the family to the mean structure are 0.67 +/- 0.12 A for the backbone and 1.00 +/- 0.12 A for all heavy atoms). Comparison of the Cu(I) and apo conformations of the protein reveals that the Cu(I) binding cysteines move from a buried site in the bound metal form to a solvent-exposed conformation on the surface of the protein after copper release. Furthermore, copper release leads to a less helical character in the metal binding site. Comparison with the Hg(II)-Atx1 solid-state structure [Rosenzweig, A. C., Huffman, D. L., Hou, M. Y., Wernimont, A. K., Pufahl, R. A., and O'Halloran, T. V. (1999) Structure 7, 605-617] provides insights into the copper transfer mechanism, and a pivotal role for Lys65 in the metal capture and release process is proposed.

Solution structure of the yeast copper transporter domain Ccc2a in the apo and Cu(I)-loaded states.

Ccc2 is an intracellular copper transporter in Saccharomyces cerevisiae and is a physiological target of the copper chaperone Atx1. Here we describe the solution structure of the first N-terminal MTCXXC metal-binding domain, Ccc2a, both in the presence and absence of Cu(I). For Cu(I)-Ccc2a, 1944 meaningful nuclear Overhauser effects were used to obtain a family of 35 structures with root mean square deviation to the average structure of 0.36 +/- 0.06 A for the backbone and 0.79 +/- 0.05 A for the heavy atoms. For apo-Ccc2a, 1970 meaningful nuclear Overhauser effects have been used with 35 (3)J(HNHalpha) to obtain a family of 35 structures with root mean square deviation to the average structure of 0.38 +/- 0.06 A for the backbone and 0.82 +/- 0.07 A for the heavy atoms. The protein exhibits a betaalphabetabetaalphabeta, ferrodoxin-like fold similar to that of its target Atx1 and that of a human counterpart, the fourth metal-binding domain of the Menkes protein. The overall fold remains unchanged upon copper loading, but the copper-binding site itself becomes less disordered. The helical context of the copper-binding site, and the copper-induced conformational changes in Ccc2a differ from those in Atx1. Ccc2a presents a conserved acidic surface which complements the basic surface of Atx1 and a hydrophobic surface. These results open new mechanistic aspects of copper transporter domains with physiological copper donor and acceptor proteins.

Reduction of enteric microorganisms at the Upper Occoquan Sewage Authority Water Reclamation Plant.

The Upper Occoquan Sewage Authority (UOSA) Water Reclamation Plant, Centreville, Virginia, is a state-of-the-art wastewater treatment plant that was created to treat area wastewater and provide protection for the Occoquan Reservoir. This study investigated UOSA's unit processes as barriers to pathogenic as well as altemative and traditional-indicator microorganisms. Samples were collected once a month for 1 year from eight sites within UOSA's advanced wastewater reclamation plant. The eight sites were monitored for indicator bacteria total and fecal coliforms, enterococci, Clostridium, coliphage (the virus that infects Escherichia coli), human enteroviruses, and enteric protozoa. Overall, the plant was able to achieve a 5- to 7-log10 reduction of bacteria, 5-log10 reduction of enteroviruses, 4-log10 reduction for Clostridium, and 4.6-log10 reduction of protozoa. Total coliforms, enterococci, Clostridium, coliphage, Cryptosporidium, and Giardia were all detected in four or fewer samples of the final effluent. No enteroviruses or fecal coliforms were detected in the final effluent. The microbiological quality of reclaimed water and the reservoir water were compared. In every case, the treated wastewater was of a better quality than the ambient water in the reservoir, thus indicating that the reclaimed water will not adversely affect the water quality for downstream users.

Three new helminth species from two endemic plethodontid salamanders, Typhlomolge rathbuni and Eurycea nana, in central Texas.

Helminthological examination of two rare, endemic species of plethodontid salamanders, the Texas blind salamander (Typhlomolge rathbuni Stejneger) and the San Marcos dwarf salamander (Eurycea nana Bishop), from the subterranean waters and springs in San Marcos, Hays County, central Texas, USA revealed the presence of three new, previously undescribed species of intestinal helminths: Brachycoelium longleyi sp. n. (Trematoda) from T. rathbuni (type host) and E. nana, Dendronucleata americana sp. n. (Acanthocephala) from T. rathbuni, and Amphibiocapillaria texensis sp. n. (Nematoda) from T. rathbuni; nematode larvae probably belonging to the last named species were recorded from E. nana. Brachycoelium longleyi can be distinguished from all congeners primarily by its conspicuously small eggs among other features, whereas A. texensis differs from its closest congeneric species A. tritonispiunctati mainly in the structure of mature eggs and a markedly shorter spicule. Dendronucleata americana is the first species of the family Dendronucleatidae from the New World, differing from its Asian congeners mainly in the number and arrangement of proboscis hooks, number of giant hypodermic nuclei and in the position of testes.

Structural basis for copper transfer by the metallochaperone for the Menkes/Wilson disease proteins.

The Hah1 metallochaperone protein is implicated in copper delivery to the Menkes and Wilson disease proteins. Hah1 and the N-termini of its target proteins belong to a family of metal binding domains characterized by a conserved MT/HCXXC sequence motif. The crystal structure of Hah1 has been determined in the presence of Cu(I), Hg(II), and Cd(II). The 1.8 A resolution structure of CuHah1 reveals a copper ion coordinated by Cys residues from two adjacent Hah1 molecules. The CuHah1 crystal structure is the first of a copper chaperone bound to copper and provides structural support for direct metal ion exchange between conserved MT/HCXXC motifs in two domains. The structures of HgHah1 and CdHah1, determined to 1.75 A resolution, also reveal metal ion coordination by two MT/HCXXC motifs. An extended hydrogen bonding network, unique to the complex of two Hah1 molecules, stabilizes the metal binding sites and suggests specific roles for several conserved residues. Taken together, the structures provide models for intermediates in metal ion transfer and suggest a detailed molecular mechanism for protein recognition and metal ion exchange between MT/HCXXC containing domains.

Energetics of copper trafficking between the Atx1 metallochaperone and the intracellular copper transporter, Ccc2.

The Atx1 metallochaperone protein is a cytoplasmic Cu(I) receptor that functions in intracellular copper trafficking pathways in plants, microbes, and humans. A key physiological partner of the Saccharomyces cerevisiae Atx1 is Ccc2, a cation transporting P-type ATPase located in secretory vesicles. Here, we show that Atx1 donates its metal ion cargo to the first N-terminal Atx1-like domain of Ccc2 in a direct and reversible manner. The thermodynamic gradient for metal transfer is shallow (K(exchange) = 1.4 +/- 0.2), establishing that vectorial delivery of copper by Atx1 is not based on a higher copper affinity of the target domain. Instead, Atx1 allows rapid metal transfer to its partner. This equilibrium is unaffected by a 50-fold excess of the Cu(I) competitor, glutathione, indicating that Atx1 also protects Cu(I) from nonspecific reactions. Mechanistically, we propose that a low activation barrier for transfer between partners results from complementary electrostatic forces that ultimately orient the metal-binding loops of Atx1 and Ccc2 for formation of copper-bridged intermediates. These thermodynamic and kinetic considerations suggest that copper trafficking proteins overcome the extraordinary copper chelation capacity of the eukaryotic cytoplasm by catalyzing the rate of copper transfer between physiological partners. In this sense, metallochaperones work like enzymes, carefully tailoring energetic barriers along specific reaction pathways but not others.

A most-probable-number assay for enumeration of infectious Cryptosporidium parvum oocysts.

Cryptosporidium is globally established as a contaminant of drinking and recreational waters. A previously described cell culture infectivity assay capable of detecting infectious oocysts was adapted to quantify viable oocysts through sporozoite invasion and clustering of foci. Eight experiments were performed by using oocysts less than 4 months of age to inoculate host HCT-8 cell monolayers. Oocysts were diluted in a standard 5- or 10-fold multiple dilution format, levels of infection and clustering were determined, and the most probable number (MPN) of infectious oocysts in the stock suspension was calculated. The MPN was compared to the initial oocyst inoculum to determine the level of correlation. For oocysts less than 30 days of age, the correlation coefficient (r) was 0.9726 (0.9306 to 0.9893; n = 20). A two-tailed P value (alpha = 0.05) indicated that P was less than 0.0001. This strong correlation suggests that the MPN can be used to effectively enumerate infectious oocysts in a cell culture system. Age affected the degree of oocyst infectivity. Oocyst infectivity was tested by the focus detection method (FDM)-MPN assay and in BALB/c mice before and after treatment with pulsed white light (PureBrite). The FDM-MPN assay and animal infectivity assays both demonstrated more than a 4 log(10) inactivation. Municipal water systems and a host of other water testing organizations could utilize the FDM-MPN assay for routine survival and disinfection studies.

Crystal structure of the Atx1 metallochaperone protein at 1.02 A resolution.

BACKGROUND: Metallochaperone proteins function in the trafficking and delivery of essential, yet potentially toxic, metal ions to distinct locations and particular proteins in eukaryotic cells. The Atx1 protein shuttles copper to the transport ATPase Ccc2 in yeast cells. Molecular mechanisms for copper delivery by Atx1 and similar human chaperones have been proposed, but detailed structural characterization is necessary to elucidate how Atx1 binds metal ions and how it might interact with Ccc2 to facilitate metal ion transfer. RESULTS: The 1.02 A resolution X-ray structure of the Hg(II) form of Atx1 (HgAtx1) reveals the overall secondary structure, the location of the metal-binding site, the detailed coordination geometry for Hg(II), and specific amino acid residues that may be important in interactions with Ccc2. Metal ion transfer experiments establish that HgAtx1 is a functional model for the Cu(I) form of Atx1 (CuAtx1). The metal-binding loop is flexible, changing conformation to form a disulfide bond in the oxidized apo form, the structure of which has been solved to 1.20 A resolution. CONCLUSIONS: The Atx1 structure represents the first structure of a metallochaperone protein, and is one of the largest unknown structures solved by direct methods. The structural features of the metal-binding site support the proposed Atx1 mechanism in which facile metal ion transfer occurs between metal-binding sites of the diffusible copper-donor and membrane-tethered copper-acceptor proteins. The Atx1 structural motif represents a prototypical metal ion trafficking unit that is likely to be employed in a variety of organisms for different metal ions.