Using experimental and computational energy equilibration to understand hierarchical self-assembly of Fmoc-dipeptide amphiphiles.

Despite progress, a fundamental understanding of the relationships between the molecular structure and self-assembly configuration of Fmoc-dipeptides is still in its infancy. In this work, we provide a combined experimental and computational approach that makes use of free energy equilibration of a number of related Fmoc-dipeptides to arrive at an atomistic model of Fmoc-threonine-phenylalanine-amide (Fmoc-TF-NH2) which forms twisted fibres. By using dynamic peptide libraries where closely related dipeptide sequences are dynamically exchanged to eventually favour the formation of the thermodynamically most stable configuration, the relative importance of C-terminus modifications (amide versus methyl ester) and contributions of aliphatic versus aromatic amino acids (phenylalanine F vs. leucine L) is determined (F > L and NH2 > OMe). The approach enables a comparative interpretation of spectroscopic data, which can then be used to aid the construction of the atomistic model of the most stable structure (Fmoc-TF-NH2). The comparison of the relative stabilities of the models using molecular dynamic simulations and the correlation with experimental data using dynamic peptide libraries and a range of spectroscopy methods (FTIR, CD, fluorescence) allow for the determination of the nanostructure with atomistic resolution. The final model obtained through this process is able to reproduce the experimentally observed formation of intertwining fibres for Fmoc-TF-NH2, providing information of the interactions involved in the hierarchical supramolecular self-assembly. The developed methodology and approach should be of general use for the characterization of supramolecular structures.

Sequence-Dependent Melting and Refolding Dynamics of RNA UNCG Tetraloops Using Temperature-Jump/Drop Infrared Spectroscopy.

Time-resolved temperature-jump/drop infrared (IR) spectroscopy has been used to measure the impact of stem base sequence on the melting and refolding dynamics of ribonucleic acid (RNA) tetraloops. A series of three 12-nucleotide RNA hairpin sequences were studied, each featuring a UACG tetraloop motif and a double-stranded stem containing four base pairs. In each case, the stem comprised three GC pairs plus a single AU base pair inserted at the closing point of the loop (RNAloop), in the middle of the stem (RNAmid), or at the stem terminus (RNAend). Results from analogous DNA tetraloop (TACG) sequences were also obtained. Inclusion of AU or AT base pairs in the stem leads to faster melting of the stem-loop structure compared to a stem sequence featuring four GC base pairs while refolding times were found to be slower, consistent with a general reduction in stem-loop stability caused by the AU/AT pair. Independent measurement of the dynamic timescales for melting and refolding of ring vibrational modes of guanine (GR) and adenine (AR) provided position-specific insight into hairpin dynamics. The GR-derived data showed that DNA sequences melted more quickly (0.5 ± 0.1 to 0.7 ± 0.1 µs at 70 °C) than analogous RNA sequences (4.3 ± 0.4 to 4.4 ± 0.3 µs at 70 °C). Position-sensitive data from the AR modes suggests that DNA hairpins begin melting from the terminal end of the stem toward the loop while RNA sequences begin melting from the loop. Refolding timescales for both RNA and DNA hairpins were found to be similar (250 ± 50 µs at 70 °C) except for RNAend and DNAloop which refolded much more slowly (746 ± 36 and 430 ± 31 µs, respectively), showing that the refolding pathway is significantly impaired by the placement of AU/AT pairs at different points in the stem. We conclude that conformational changes of analogous pairs of RNA and DNA tetraloops proceed by different mechanisms.

Measuring RNA UNCG Tetraloop Refolding Dynamics Using Temperature-Jump/Drop Infrared Spectroscopy.

Determining the structural dynamics of RNA and DNA is essential to understanding their cellular function, but direct measurement of strand association or folding remains experimentally challenging. Here we illustrate a temperature-jump/drop method able to reveal refolding dynamics. Time-resolved temperature-jump/drop infrared spectroscopy is used to measure the melting and refolding dynamics of a 12-nucleotide RNA sequence comprising a UACG tetraloop and a four-base-pair double-stranded GC stem, comparing them to an equivalent DNA (TACG) sequence. Stem-loop melting occurred an order of magnitude more slowly in RNA than DNA (6.0 ± 0.1 µs versus 0.8 ± 0.1 µs at 70 °C). In contrast, the refolding dynamics of both sequences occurred on similar time scales (200 µs). While the melting and refolding dynamics of RNA and DNA hairpins both followed Arrhenius temperature dependences, refolding was characterized by an apparent negative activation energy, consistent with a mechanism involving multiple misfolded intermediates prior to zipping of the stem base pairs.

Structure and vibrational dynamics of model compounds of the [FeFe]-hydrogenase enzyme system via ultrafast two-dimensional infrared spectroscopy.

Ultrafast two-dimensional infrared (2D) spectroscopy has been applied to study the structure and vibrational dynamics of (mu-S(CH2)3S)Fe2(CO)6, a model compound of the active site of the [FeFe]-hydrogenase enzyme system. Comparison of 2D-IR spectra of (mu-S(CH2)3S)Fe2(CO)6 with density functional theory calculations has determined that the solution-phase structure of this molecule is similar to that observed in the crystalline phase and in good agreement with gas-phase simulations. In addition, vibrational coupling and rapid (<5 ps) solvent-mediated equilibration of energy between vibrationally excited states of the carbonyl ligands of the di-iron-based active site model are observed prior to slower (approximately 100 ps) relaxation to the ground state. These dynamics are shown to be solvent-dependent and form a basis for the future determination of the vibrational interactions between active site and protein.

Australian-Indonesian collaboration in veterinary arbovirology--a review.

Australian-Indonesian collaboration in veterinary development programs has led to significant advances in the study of arboviruses. This paper reviews the resulting knowledge of arboviral infections of livestock in Indonesia. The first recognized arboviral disease of animals in Indonesia was bovine ephemeral fever. Serology indicates that the virus is widespread, as are related rhabdoviruses. Local sheep appear resistant to bluetongue disease, but imported sheep have suffered mortalities. Bluetongue viral serotypes 1, 7, 9, 12, 21 and 23 have been isolated from sentinel cattle; 1, 21 and 23 at widely separate locations. Bluetongue serotype 21 has been isolated from Culicoides spp. Serological reactors to Akabane virus are widespread, as are reactors to the flavivirus group. Japanese encephalitis, isolated from sentinel pigs, is the flavivirus of most veterinary importance but the limit of its easterly distribution is unknown. Many of the arboviruses present in Indonesia are also present in Australia and elsewhere in Asia. Their patterns of mobility among countries in the region are largely undescribed, but there are opportunities for further regional collaboration.

EHDV-1, a new Australian serotype of epizootic haemorrhagic disease virus isolated from sentinel cattle in the Northern Territory.

In 1992, a virus (DPP2209) isolated from sentinel cattle located at Coastal Plains Research Station, latitude 12 degrees 39'S, longitude 131 degrees 20'E, approximately 60 km east of Darwin, Northern Territory. This virus was identified as a serotype of epizootic haemorrhagic disease (EHD) of deer virus previously undescribed in Australia. An additional 17 isolation of this virus were made from eight animals during the period February to May. Electron microscopic studies showed the presence of orbivirus-like structures. Serogrouping ELISA, indirect immunofluorescence assay and the serogrouping plaque reduction neutralisation test indicated the virus was a member of the epizootic haemorrhagic disease serogroup. Serotype specific plaque reduction neutralisation tests, indicated the virus was a member of the epizootic haemorrhagic disease serogroup not previously isolated in Australia. Analysis of the VP3 gene confirmed this observation. Cross neutralisation testing of the isolate with known epizootic haemorrhagic disease serotype viruses including endemic Australian and exotic strains identified isolate DPP2209 as epizootic haemorrhagic disease virus serotype 1.

Transient 2D-IR spectroscopy of inorganic excited states.

Time-resolved infrared spectroscopy has been proven to be a powerful tool for investigating the structure, dynamics and reactivity of electronically-excited states of inorganic molecules. As applications drive the production of ever more complex molecules however, experimental tools that can deliver more detailed spectroscopic information, or separate multiple contributions to complex signals will become increasingly valuable. In this Perspective, the extension of ultrafast infrared spectroscopy of inorganic excited states to a second frequency dimension using transient 2D-IR spectroscopy (T-2D-IR) methods is discussed. Following a brief discussion of the experimental methodologies, examples will be given of applications of T-2D-IR ranging from studies of the spectroscopy, structure and dynamics of photochemical intermediates to new tools for correlating vibrational modes in ground and excited electronic states and the investigation of excited state solvation dynamics. Future directions for these experiments are also discussed.

Temporal activity of biting midges (Diptera: Ceratopogonidae) on cattle near Darwin, Northern Territory, Australia.

The activity of nine species of biting midges aspirated from cattle was recorded in the late afternoon, evening and early morning at a site near Darwin, Northern Territory, between March and June in 1999 and 2001. There were no significant differences between the temporal activity patterns for nulliparous and parous females of any species. Nulliparous females dominated collections of all species except Culicoides marksi. C. actoni and Forcipomyia (Lasiohelea) sp., were mostly active during daylight hours while C. peregrinus, C. bundyensis and C. brevipalpis, were nocturnal. Differences in the peak activity of C. brevitarsis were noted between years and occurred slightly earlier than that observed at other sites. C. fulvus, C. marksi and C. oxystoma were generally crepuscular but differed in the length and peak period of activity. C. actoni was four times more active in the evening than in the morning while C. marksi and C. peregrinus, were respectively 2.6 and 3.4 times more active in the morning than in the evening. Numbers of the other six species were not significantly different in the evening and morning. All nine species were collected at least once from cattle shortly after dawn.

Bluetongue virus does not persist in naturally infected cattle.

Studies were designed to test if observations by Takamatsu et al. in 2003 were applicable to natural infection of cattle with bluetongue virus (BTV). These observations suggested that ovine gamma delta T-cells could become persistently infected and subsequent midge feeding could induce virus replication. Skin biopsies and blood were collected from 28 cattle naturally infected with BTV-1. Blood samples were processed for virus isolation by embryonated chicken egg inoculation and for serology by BTV competitive enzyme-linked immunosorbent assay and BTV-1 virus neutralisation. BTV-1 was isolated from the blood of all animals and serology confirmed infection with BTV-1. A total of 288 skin biopsies were collected and cultured in the presence of interleukin 2 and epidermal growth factor. Sampling commenced as soon as either serology or virus isolation indicated infection with BTV and continued at weekly intervals for at least eight weeks then monthly for another two months. The natural viraemias in this experiment ranged from one to five weeks. BTV-1 was isolated from only one skin biopsy sample. This sample was collected during the week in which the animal was viraemic. These findings provide compelling evidence that BTV does not persist in gamma delta T-cells in the skin of naturally infected cattle.

Monitoring of acidified milk gel formation by ultrasonic shear wave measurements. High-frequency viscoelastic moduli of milk and acidified milk gel.

In the present paper we describe the application of the thickness shear mode resonator technique for the measurement of viscoelastic parameters of milk and acidified milk gels in the megahertz frequency range. The technique provides information on the viscoelasticity of milk and milk gels in the time scale of 10(-8) to 10(-7) s. The length scale of the measurements, determined by the depth of penetration and the wavelength of the shear wave, falls between the submicron and micron range. In milk acidified by glucono-delta-lactone we observed an increase in the high- and low-frequency loss modulus, G", below pH 5.1, indicating aggregation of casein particles into clusters. There was a sharp rise in high- and low-frequency storage (G') and loss (G") moduli between pH 4.85 and 4.7, as a result of gel network formation in acidified milk. Both G" and G' of milk gel in the megahertz frequency range are several orders higher than those we obtained at low frequencies (0.02 to 10 Hz) using dynamic rheology. The high-frequency (5 to 25 MHz) viscosity of milk was found to be the same as at low frequencies. Overall, our results demonstrate the high sensitivity of the ultrasonic shear wave measurements to the changes in the rheological parameters in acidified milk during gelation.

Protection of cattle from Culicoides spp. in Australia by shelter and chemical treatments.

Trials were conducted in three regions of Australia to investigate the potential for improvised shelters and chemical treatments to reduce feeding by Culicoides on cattle and thereby minimise the risk of bluetongue transmission during transport of cattle to ports. Various designs and combinations of roofs and walls were placed around penned cattle. Chemical treatments were applied to other penned cattle. Culicoides were collected from the cattle by vacuum samplers or by light traps in the pens. Roofs alone did not consistently reduce the numbers of Culicoides brevitarsis or C. fulvus and increased the numbers of C. actoni collected. Walls alone reduced the numbers of C. wadai but not C. brevitarsis. Roofs and walls in combination reduced the numbers of C. brevitarsis and C. wadai. The chemical treatments 'Flyaway' (a blend of repellents) and fenvalerate reduced the numbers of C. brevitarsis and C. wadai up to 52 h post treatment.

Excretion of bluetongue virus in cattle semen: a feature of laboratory-adapted virus.

A series of experiments was conducted over a period of four years and involved both young (2-4 years) and old bulls (5-15 years) that were both naturally and experimentally infected with bluetongue virus (BTV). Several different virus serotypes were studied. In the Northern Territory, young bulls were exposed to natural infection with BTV over three wet seasons. During this time, bulls were infected with BTV-1, BTV-3, BTV-16 and BTV-20. In New South Wales, semen samples were examined from a large group of bulls of mixed ages that were naturally infected with BTV-1. Experimental infections in both young and old bulls (5-8 animals per group) employed both 'wild-type' and laboratory-adapted viruses from serotypes 1 and 23. A total of 41 bulls were included in the studies of natural BTV infection and 52 bulls in experimental infections. There was no evidence of BTV in any of the semen samples collected from naturally infected bulls or experimentally infected young bulls. BTV was detected intermittently in semen from a number of old bulls infected with both laboratory-adapted BTV-1 and BTV-23. These detections occurred during or immediately after the period of detectable viraemia. Virus was also detected in a few semen samples from very old bulls infected with 'wild-type' BTV-23. These samples were collected during the period of viraemia and there was usually evidence of blood in the semen. Viraemia varied in duration between 17 and 38 days. Following immunosuppression, there was no evidence of resurgence of viraemia, or excretion of virus in semen, even in animals in which virus had been previously detected in semen. When the bulls were slaughtered, virus was not detected in any tissues.

Studies on the epidemiology of bluetongue virus in China.

Sentinel herds of large ruminants were established at five centres in Yunnan Province, Peoples Republic of China, between 1995 and 1997. The application of a sensitive antigen capture ELISA to facilitate virus isolation procedures led to the isolation of 108 strains of bluetongue (BLU) virus. Serotypes isolated included types 1, 2, 3, 4, 9, 11, 12, 15, 16, 21 and 23. Virus transmission occurred over a period of 1-3 months at each of the four positive sites, giving an overall BLU virus transmission period for the province of 5 months, from early June to early November. The greatest level of transmission took place in July and August. The duration of viraemia in individual animals varied from 1 to 7 weeks, with a mean calculated for each serotype between 6 and 20 days. The study represents the first detailed investigation of the epidemiology of BLU in China utilizing sentinel herds.