Application of the Droplet Digital Polymerase Chain Reaction (ddPCR) Platform for Detection and Quantification of Vertebrate Host DNA in Engorged Mosquitoes.

Hematophagous arthropod bloodmeal identification has remained a challenge in the field of vector biology, but these studies are important to understand blood feeding patterns of arthropods, spatial, and temporal patterns in arbovirus transmission cycles, and risk of human and veterinary disease. We investigated the use of an existing vertebrate primer set for use on the droplet digital polymerase chain reaction (ddPCR) platform, to explore the use of this technology in the identification and quantification of vertebrate DNA in mosquito blood meals. Host DNA was detectable 48-h post-engorgement in some mosquitoes by ddPCR, compared with 24-h post-engorgement using traditional PCR. The capability of ddPCR for absolute quantification of template DNA offers unique potential applications of this new technology to field studies on the ecology of vector-borne diseases, but currently with limited scope.

Design and construction of a single tube, quantitative endpoint, LATE-PCR multiplex assay for ventilator-associated pneumonia.

AIMS: The goal of this study was to develop a molecular diagnostic multiplex assay for the quantitative detection of microbial pathogens commonly responsible for ventilator-associated pneumonia (VAP) and their antibiotic resistance using linear-after-the-exponential polymerase chain reaction (LATE-PCR). METHOD AND RESULTS: This multiplex assay was designed for the quantitative detection and identification of pathogen genomic DNA of methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii, Pseudomonas aeruginosa, plus a control target from Lactococcus lactis. After amplification, the single-stranded amplicons were detected simultaneously in the same closed tube by hybridization to low-temperature molecular beacon probes labelled with four differently coloured fluorophores. The resulting hybrids were then analysed by determining the fluorescence intensity of each of the four fluorophores as a function of temperature. CONCLUSIONS: This LATE-PCR single tube multiplex assay generated endpoint fluorescent contours that allowed identification of all microbial pathogens commonly responsible for VAP, including MRSA. The assay was quantitative, identifying the pathogens present in the sample, no matter whether there were as few as 10 or as many 100 000 target genomes. SIGNIFICANCE AND IMPACT OF THE STUDY: This assay is rapid, reliable and sensitive and is ready for preclinical testing using samples recovered from patients suffering from ventilator-associated pneumonia.

Design of a single-tube, endpoint, linear-after-the-exponential-PCR assay for 17 pathogens associated with sepsis.

AIMS: The goal of this study was to construct a single-tube multiplex molecular diagnostic assay using linear-after-the-exponential (LATE)-PCR for the detection of 17 microbial pathogens commonly associated with septicaemia. METHODS AND RESULTS: The assay described here detects 17 pathogens associated with sepsis via amplification and analysis of gene-specific sequences. The pathogens and their targeted genes were: Klebsiella spp. (phoE); Acinetobacter baumannii (gyrB); Staphylococcus aureus (spa); Enterobacter spp. (thdF); Pseudomonas aeruginosa (toxA); coagulase-negative staphylococci (tuf), Enterococcus spp. (tuf); Candida spp. (P450). A sequence from an unidentified gene in Lactococcus lactis, served as a positive control for assay function. LATE-PCR was used to generate single-stranded amplicons that were analysed at endpoint over a wide range of temperatures in four fluorescent colours. Each target was detected by its pattern of hybridization to a sequence-specific low-temperature fluorescent probe derived from molecular beacons. CONCLUSIONS: All 17 microbial targets were detected in samples containing low numbers of pathogen genomes in the presence of high levels of human genomic DNA. SIGNIFICANCE AND IMPACT OF THE STUDY: This assay used new technology to achieve an advance in the field of molecular diagnostics: a single-tube assay for detection of pathogens commonly responsible for septicaemia.

Verification of monoplex and multiplex linear-after-the-exponential PCR gene-specific sepsis assays using clinical isolates.

AIMS: To verify monoplex and multiplex gene-specific linear-after-the-exponential polymerase chain reaction (LATE-PCR) assays for identifying 17 microbial pathogens (i.e., Klebsiella sp., Acinetobacter baumannii, Staphylococcus aureus, Enterobacter sp., Pseudomonas aeruginosa, coagulase negative staphylococci, Enterococcus sp., Candida sp.) commonly associated with septicaemia using clinical isolates. METHODS AND RESULTS: Clinical isolates of each target pathogen were collected from the University of California, Davis Medical Center (UCDMC) microbiology laboratory. Five microlitres (µl) of each culture suspension (1 × 10(8) CFU ml(-1) ) were added to 20 µl of monoplex mastermix. DNA extracted from clinical isolates was tested in multiplex. Monoplex assays demonstrated 100% sensitivity at this input level, except Enterobacter cloacae (2.7%), Ac. baumannii (57%) and Ps. aeruginosa (97.8%). All clinical isolates were positive in multiplex, with the exception of two Ac. baumannii, two Klebsiella oxytoca and two Candida parapsilosis isolates. CONCLUSIONS: Sixteen pathogens can be identified by monoplex LATE-PCR assays with sensitivities ≥ 97.8%. The multiplex assay demonstrated 91.4% sensitivity when tested with DNA extracted from 70 different target strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the potential of LATE-PCR to serve as an adjunct to culture if the reagents are optimized for sensitivity. Results warrant further testing through analytical and clinical validation of the multiplex assay.

Single-wavelength anomalous diffraction phasing revisited.

Multiwavelength anomalous diffraction (MAD) phasing has become a routinely used tool for determining new macromolecular structures. The MAD method has stringent data-collection requirements, typically necessitating radiation-resistant crystals and access to a tunable synchrotron beamline. In cases where synchrotron time, monochromator tunability or radiation damage is a concern or where high-throughput structure determination is desired, phasing methods capable of producing interpretable electron-density maps from less data become attractive alternatives to MAD. The increasing availability of tunable synchrotron data-collection facilities prompted the authors to revisit single-wavelength anomalous diffraction (SAD) phasing used in conjunction with a phase-ambiguity resolving method such as solvent flattening. The anomalous diffraction from seven different selenomethionine-labelled protein crystals has been analysed and it is shown that in conjunction with solvent flattening, diffraction data from the peak anomalous wavelength alone can produce interpretable electron-density maps of comparable quality to those resulting from full MAD phasing. Single-wavelength anomalous diffraction (SAD) phasing can therefore be a time-efficient alternative to MAD. The data also show that radiation damage can have a significant effect on the quality of SAD/MAD diffraction data. These results may be useful in the design of optimal strategies for collection of the diffraction data.

Crystal structure of the vesicular transport protein Sec17: implications for SNAP function in SNARE complex disassembly.

SNAP proteins play an essential role in membrane trafficking in eukaryotic cells. They activate and recycle SNARE proteins by serving as adaptors between SNAREs and the cytosolic chaperone NSF. We have determined the crystal structure of Sec17, the yeast homolog of alpha-SNAP, to 2.9 A resolution. Sec17 is composed of an N-terminal twisted sheet of alpha-helical hairpins and a C-terminal alpha-helical bundle. The N-terminal sheet has local similarity to the tetratricopeptide repeats from protein phosphatase 5 but has a different overall twist. Sec17 also shares structural features with HEAT and clathrin heavy chain repeats. Possible models of SNAP:SNARE binding suggest that SNAPs may function as lever arms, transmitting forces generated by conformational changes in NSF/Sec18 to drive disassembly of SNARE complexes.

Long-term variations in cyclic light intensity and dietary vitamin A intake modulate lipofuscin content of the retinal pigment epithelium.

Experiments were conducted to determine whether the intensity of cyclic light exposure to the retina over a long period of time affects retinoid-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE). Albino rats were maintained from weaning on diets either containing (+A) or lacking (-A) retinyl palmitate, which can be metabolized to the retinoids involved in the visual cycle. Animals in each dietary group were divided between bright (L) and dim (D) cyclic light treatments. Thus, the experiments employed the following four treatment groups: +A/D, +A/L, -A/D, and -A/L. After 6, 12, and 15 months from the start of the treatments, animals in each group were killed for quantitative determination of: 1) retinal photoreceptor densities; 2) RPE lipofuscin content; and 3) RPE lipofuscin fluorescence intensity. Animals in the L groups had a lower volume of RPE lipofuscin than those in the D groups fed the same diet. Among the -A rats, this reduced lipofuscin volume could be attributed to a light-enhanced depletion of vitamin A from the retina and an accompanying loss of photoreceptor cells. In the +A animals, however, there were no differences in photoreceptor densities between the D and L groups. In the -A rats, the volume of RPE lipofuscin decreased between 6 and 15 months of age, whereas it increased in the +A animals. In contrast, lipofuscin fluorescence intensity increased between 6 and 15 months of age in all four treatment groups. However, in the +A rats, the fluorescence intensity was lower in the L than in the D group at all three ages. In the -A groups, light level had no effect on lipofuscin fluorescence intensity. At all three ages, fluorescence intensity was lower in the -A animals than in +A rats. Thus, at light intensities below those that induce acute retinal degeneration, long-term exposure to higher intensity light inhibits the age-related increase in RPE lipofuscin volume. A decrease in the volume of RPE lipofuscin after the retina is depleted of vitamin A suggests that lipofuscin is turned over, and that RPE lipofuscin content is determined by a balance between the rates at which lipofuscin is formed and at which it is eliminated from the RPE. An age-related increase in lipofuscin-specific fluorescence intensity after vitamin A depletion from the retina suggests that lipofuscin fluorophores may continue to form slowly from retinoids that have been modified such that they can no longer enter the visual cycle.

Experimentally based orientational refinement of membrane protein models: A structure for the Influenza A M2 H+ channel.

The 97-residue M2 protein from Influenza A virus forms H+-selective ion channels which can be attributed solely to the homo-tetrameric alpha-helical transmembrane domain. Site-directed infrared dichroism spectra were obtained for the transmembrane domain of M2, reconstituted in lipid vesicles. Data analysis yielded the helix tilt angle beta=31.6(+/-6.2) degrees and the rotational pitch angle about the helix axis for residue Ala29 omegaAla29=-59.8(+/-9.9) degrees, whereby omega is defined as zero for a residue located in the direction of the helix tilt. A structure was obtained from an exhaustive molecular dynamics global search protocol in which the orientational data are utilised directly as an unbiased refinement energy term. Orientational refinement not only allowed selection of a unique structure but could also be shown to increase the convergence towards that structure during the molecular dynamics procedure. Encouragingly, the structure obtained is highly consistent with all available mutagenesis and conductivity data and offers a direct chemical insight that relates the altered functionality of the channel to its structure.

Folding intermediates of SNARE complex assembly.

SNARE (soluble NSF attachment protein receptor) proteins assemble into a stable complex essential for vesicle-membrane fusion. To further understand SNARE function we have used solution nuclear magnetic resonance (NMR) spectroscopy to characterize three assembly states of a yeast SNARE complex: first, the 'closed' conformation of Sso1; second, the binary complex of Sso1 and Sec9; and third, the ternary complex of Sso1, Sec9 and Snc1. Sec9 and Snc1 are unstructured in isolation. Sso1 likely consists of a four helix bundle formed by part of the C-terminal Hcore domain and the N-terminal H(A)H(B)H(C) domain, and this bundle is flanked on both sides by large flexible regions. Sso1 switches to an 'open' state when its Hcore domain binds Sec9. Conformational switching of the Hcore domain, via H(A)H(B)H(C), may provide a key regulatory mechanism in SNARE assembly. Formation of binary and ternary complexes induces additional alpha-helical structure in previously unstructured regions. Our data suggest a directed assembly process beginning distal to the membrane surfaces and proceeding toward them, bringing membranes into close proximity and possibly leading to membrane fusion.

Reversible accumulation of lipofuscin-like inclusions in the retinal pigment epithelium.

PURPOSE: The amounts of autofluorescent lysosomal storage bodies, known as lipofuscin, increase during senescence in the retinal pigment epithelia (RPEs) of mammalian eyes. This increase in lipofuscin content may result from a failure of the RPE to dispose of any lipofuscin constituents once they have formed. Alternatively, the RPE may eliminate lipofuscin but at a rate insufficient to prevent its accumulation. Experiments were conducted to distinguish between these two possibilities. METHODS: Albino rats were given intravitreal injections of the protease inhibitor leupeptin, which induces a rapid accumulation of lipofuscin-like inclusions in the RPE. The amount of these inclusions in the RPE was monitored as a function of time after the leupeptin treatment with quantitative ultrastructural analysis. In addition, the intensity of lipofuscin-specific fluorescence in the RPE was monitored over the same time period with the use of quantitative microfluorometry. These parameters were also followed in untreated control eyes of age-matched animals. RESULTS: A single leupeptin injection resulted in a rapid massive accumulation of electron-dense inclusion bodies in the RPE. These inclusions appeared to be derived primarily from phagocytosed photoreceptor outer segments. Accompanying the accumulation of these inclusions was a significant increase in lipofuscin-specific fluorescence in the RPE. Over a 12-week period after the leupeptin treatment, the amounts of inclusion material and the fluorescence intensities returned to normal levels. CONCLUSIONS: These findings suggest that the age-related increase in RPE lipofuscin content results from an imbalance in the rates of lipofuscin formation and disposal rather than from a complete absence of a disposal mechanism. The results imply that turnover of lipofuscin constituents may be rapid relative to the animals' life span. Thus, it may be possible to slow or reverse the age-related increase in RPE lipofuscin content by either inhibiting the processes involved in lipofuscin formation or enhancing the disposal processes.

Recent developments for the efficient crystallographic refinement of macromolecular structures.

Macromolecular crystallographic refinement has recently been made more efficient by the use of cross-validated maximum likelihood targets and torsion-angle molecular dynamics simulated annealing. In combination with automated model building methods, the amount of manual intervention required to complete and refine a structure is greatly reduced.

Crystallography & NMR system: A new software suite for macromolecular structure determination.

A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs, the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible. The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular structure determination by X-ray crystallography and solution NMR.

Formation of a yeast SNARE complex is accompanied by significant structural changes.

The evolutionarily conserved SNARE (SNAP receptor) proteins and their complexes are key players in the docking and fusion of secretory vesicles with their target membrane. Biophysical techniques were used to characterize structural and energetic properties of the cytoplasmic domains of the yeast SNAREs Snc1 and Sso1, of the SNAP-25-like domain of Sec9, and of the Sso1:Sec9 and Sso1:Sec9:Snc1 complexes. Individually, all three SNAREs are monomeric; Sso1 shows significant secondary structure while Snc1 and Sec9 are largely unstructured. Ternary SNARE complex formation (KD <50 nM) is accompanied by a more than two-fold increase in secondary structure. This binding induced structure, the large increase in thermal stability, and the self-association of the ternary complex represent conserved properties of SNAREs that are probably important in vesicle docking and fusion.

Dietary carnitine supplements slow disease progression in a putative mouse model for hereditary ceroid-lipofuscinosis.

The childhood ceroid-lipofuscinoses are a group of autosomal recessively inherited disorders characterized by massive accumulation of autofluorescent lysosomal storage bodies in neurons as well as other cell types. The storage body accumulation is accompanied by severe degeneration of the central nervous system that results in blindness, cognitive and psychomotor degeneration, and premature death. On the basis of pathologic and biochemical criteria, a hereditary disease in the mnd mouse strain has been proposed as a model for certain types of human ceroid-lipofuscinosis. Experimental evidence suggests that the storage body accumulation in humans with juvenile and late-infantile ceroid-lipofuscinosis is linked to altered carnitine biosynthesis. On the basis of the latter observation, a study was performed to determine whether dietary carnitine supplements could slow the disease progression in the mnd mouse model. Carnitine supplementation begun at 4 weeks of age did not slow the retinal degeneration that is characteristic of this disease. It did, however, significantly elevate brain carnitine levels, slow the accumulation of autofluorescent storage bodies in brain neurons, and prolong the lifespans of the treated animals. These findings suggest that there is a link between carnitine biosynthesis and the disease pathology and indicate that carnitine supplementation may be beneficial in slowing the disease progression in humans with certain types of hereditary ceroid-lipofuscinosis.

Analysis of a yeast SNARE complex reveals remarkable similarity to the neuronal SNARE complex and a novel function for the C terminus of the SNAP-25 homolog, Sec9.

SNARE proteins represent a family of related proteins that are thought to have a central role in vesicle targeting and fusion in all eukaryotic cells. The binding properties of the neuronal proteins synaptobrevin 1 (VAMP1), syntaxin 1, SNAP-25, and soluble N-ethylmaleimide-sensitive factor attachment protein (alpha-SNAP), have been extensively studied. We report here the first biochemical characterization of a nonneuronal SNARE complex using recombinant forms of the yeast exocytic SNARE proteins Snc1, Sso1, and Sec9 and the yeast alpha-SNAP homolog, Sec17. Despite the low level of sequence identity, the association properties of the yeast and neuronal complexes are remarkably similar. The most striking difference we have found between the yeast and neuronal proteins is that individually neither of the target membrane SNAREs (t-SNAREs), Sso1 nor Sec9, show any detectable binding to the synaptobrevin homolog, Snc1. However, as a hetero-oligomeric complex, Sec9 and Sso1 show strong binding to Snc1. The clear dependence on the Sso1-Sec9 complex for t-SNARE function suggests that regulating the formation of this complex may be a key step in determining the site of vesicle fusion. In addition, we have used this in vitro assay to examine the biochemical effects of several mutations in Sec9 that result in pronounced growth defects in vivo. As expected, a temperature-sensitive mutation in the region most highly conserved between Sec9 and SNAP-25 is severely diminished in its ability to bind Sso1 and Snc1 in vitro. In contrast, a temperature-sensitive mutation near the C terminus of Sec9 shows no defect in SNARE binding in vitro. Similarly, a deletion of the C-terminal 17 residues, which is lethal in vivo, also binds Sso1 and Snc1 normally in vitro. Interestingly, we find that these same two C-terminal mutants, but not mutants that show SNARE assembly defects in vitro, act as potent dominant negative alleles when expressed behind a strong regulated promoter. Taken together these results suggest that the C-terminal domain of Sec9 is specifically required for a novel interaction that is required at a step following SNARE assembly.

Crystal structure of the two RNA binding domains of human hnRNP A1 at 1.75 A resolution.

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is an abundant eukaryotic nuclear RNA binding protein. A1 is involved in the packaging of pre-mRNA into hnRNP particles, transport of poly A+ mRNA from the nucleus to the cytoplasm and may modulate splice site selection. The crystal structure of A1(RBD1,2) reveals two independently-folded RNA binding domains (RBDs) connected by a flexible linker. Both RBDs are structurally homologous to the U1A(RBD1), and have their RNA binding platforms oriented in an anti-parallel fashion. The anti-parallel arrangement of the A1 RNA binding platforms suggests mechanisms for RNA condensation and ways of bringing together distant RNA sequences for RNA metabolism such as splicing or transport.

Torsion-angle molecular dynamics as a new efficient tool for NMR structure calculation.

Molecular dynamics in torsion-angle space was applied to nuclear magnetic resonance structure calculation using nuclear Overhauser effect-derived distances and J-coupling-constant-derived dihedral angle restraints. Compared to two other commonly used algorithms, molecular dynamics in Cartesian space and metric-matrix geometry combined with Cartesian molecular dynamics, the method shows increased computational efficiency and success rate for large proteins, and it shows a dramatically increased radius of convergence for DNA. The torsion-angle molecular dynamics algorithm starts from an extended strand conformation and proceeds in four stages: high-temperature torsion-angle molecular dynamics, slow-cooling torsion-angle molecular dynamics, Cartesian molecular dynamics, and minimization. Tests were carried out using experimental NMR data for protein G, interleukin-8, villin 14T, and a 12 base-pair duplex of DNA, and simulated NMR data for bovine pancreatic trypsin inhibitor. For villin 14T , a monomer consisting of 126 residues, structure determination by torsion-angle molecular dynamics has a success rate of 85%, a more than twofold improvement over other methods. In the case of the 12 base-pair DNA duplex, torsion-angle molecular dynamics had a success rate of 52% while Cartesian molecular dynamics and metric-matrix distance geometry always failed.