Background

Clinicians use several measures to estimate adiposity. Body mass index (BMI), although not a measure of adiposity, is commonly used to define weight status. Percent body fat (%BF) measures total body fatness, which is composed of central and peripheral fat, estimated by waist circumference (WC) and skinfold thickness, respectively. Abnormal increases in fat during puberty may reflect an increased risk of developing cardiovascular disease. Therefore, it is important to establish the normal patterns of change in clinically relevant measures of adiposity.

Purpose

To describe the normal patterns of change in clinical measures of adiposity during puberty.

Design/Methods

Multilevel modeling and linear regression analyses of 642 children in Project HeartBeat!, aged 8–18 years (non-black and black), who had assessments of BMI, %BF, WC, sums of 2- and 6-skinfolds, and pubertal stage (PS) triennially between 1991 and 1995.

Results

In males, the normal pattern from PS1 to PS5 is for %BF to decrease, skinfold thickness to remain stable, and WC to increase. However, after adjusting for height, WC does not change. In females, %BFremains stable from PS1 to PS5, whereas skin fold thickness increases. As in males waist-height ratio does not change, indicating that central adiposity does not normally increase during puberty. Although BMI increases in both genders and races from PS1 to PS5, mean values at PS5 were well below 25 kg/m2.

Conclusions

During puberty, increase in %BF is abnormal in females and even more so in males. Likewise, increase in waist-height ratio is also abnormal and may suggest an increased risk for adiposity-associated morbidity.

Two microparticle systems containing disodium cromoglycate (DSCG) alone or with polyvinyl alcohol (DSCG/PVA) were produced via spray drying and compared in terms of their physicochemical characteristics, aerosol performance and drug uptake across a pulmonary epithelial cell line (Calu-3), cultured under air interface conditions. The particle size distribution of DSCG and DSCG/PVA were similar, of spherical geometry, amorphous and suitable for inhalation purposes. Aerosolisation studies using a modified twin-stage impinger showed the DSCG/PVA to have greater aerosol performance than that of DSCG alone. Aerosol particles of DSCG and DSCG/PVA were deposited onto the surface of the Calu-3 air interface epithelium monolayer and the drug uptake from apical to basal directions measured over time. Drug uptake was measured across a range of doses to allow comparison of equivalent drug and powder mass deposition. Analysis of the data indicated that the percentage cumulative drug uptake was independent of the mass of powder deposited, but dependent on the formulation. Specifically, with the formulation containing DSCG, the diffusion rate was observed to change with respect to time (indicative of a concentration-dependent diffusion process), whilst DSCG/PVA showed a time-independent drug uptake (suggesting a zero-order depot release).

The microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper transcription factor which regulates melanocyte development and the biosynthetic melanin pathway. A notable relationship has been described between non-truncating mutations of its basic domain and Tietz syndrome, which is characterized by albinoid-like hypopigmentation of the skin and hair, rather than the patchy depigmentation seen in Waardenburg syndrome, and severe hearing loss. Twelve patients with new or recurrent non-truncating mutations of the MITF basic domain from six families were enrolled in this study. We observed a wide range of phenotypes and some unexpected features. The patients all had blue irides and pigmentation abnormalities that ranged from diffuse hypopigmentation to Waardenburg-like patches. In addition, they showed congenital complete hearing loss, diffuse hypopigmentation of the skin, freckling and ocular abnormalities, more frequently than patients with MITF mutations outside the basic domain. In conclusion, the non-truncating mutations of the basic domain do not always lead to Tietz syndrome but rather to a large range of phenotypes. Sun-exposed freckles are interestingly observed more frequently in Asian populations. This variability argues for the possible interaction with modifier loci.

Objective

To investigate the cardioprotective efficacy of remote ischaemic preconditioning (RIPC) in cardiac surgery.

Design

We have performed a systematic search of MEDLINE, EMBASE and Cochrane Central Register of Controlled Trials to identify randomized controlled trials involving RIPC.

Setting

Randomized controlled trials of RIPC in open cardiac surgery patients.

Main outcome measures

Meta-analysis was performed with the primary outcome the standardized mean difference between intervention and control groups in 12 hour postoperative troponin concentration. Heterogeneity was examined by fixed effects meta-regression.

Results

Ten studies with a total of 693 participants were included in the meta-analysis. RIPC reduced troponin levels 12 hours after surgery compared with control. The fixed and random effects differences were 0.35 (95% CI 0.19 to 0.51) and 0.53 (95% CI 0.18-0.88) respectively. However, important heterogeneity was present. Fixed effects meta-regression partially accounted for heterogeneity based on whether studies had full blinding, comprising blinding of patients, surgeons, anaesthetists and investigators. Studies with incomplete or no blinding demonstrated a larger estimate of effect, 0.74 (95% CI 0.47 to 1.00) compared to those with full blinding, 0.13 (95% CI - 0.07 to 0.33).

Conclusions

Although our analysis suggests RIPC may result in cardiac protection during cardiac surgery, the effect was most marked in studies without full blinding, with a smaller and statistically non-significant effect in fully blinded studies. We propose that further double blind randomized controlled trials investigating the cardioprotective effects of RIPC in cardiac surgery are required to resolve the current clinical uncertainty.

The CCP4 template-restraint library defines restraints for biopolymers, their modifications and ligands that are used in macromolecular structure refinement. JLigand is a graphical editor for generating descriptions of new ligands and covalent linkages.

Biological macromolecules are polymers and therefore the restraints for macromolecular refinement can be subdivided into two sets: restraints that are applied to atoms that all belong to the same monomer and restraints that are associated with the covalent bonds between monomers. The CCP4 template-restraint library contains three types of data entries defining template restraints: descriptions of monomers and their modifications, both used for intramonomer restraints, and descriptions of links for intermonomer restraints. The library provides generic descriptions of modifications and links for protein, DNA and RNA chains, and for some post-translational modifications including glycosylation. Structure-specific template restraints can be defined in a user’s additional restraint library. Here, JLigand, a new CCP4 graphical interface to LibCheck and REFMAC that has been developed to manage the user’s library and generate new monomer entries is described, as well as new entries for links and associated modifications.

The minor pilin FctB from S. pyogenes strain 90/306S was expressed in E. coli, purified and crystallized. The hexagonal FctB crystals diffracted to 2.9 Å resolution.

The minor pilin FctB is an integral part of the pilus assembly expressed by Streptococcus pyogenes. Since it is located at the cell wall, it can be hypothesized that it functions as a cell-wall anchor for the streptococcal pilus. In order to elucidate its structure, the genes for FctB from the S. pyogenes strains 90/306S and SF370 were cloned for overexpression in Escherichia coli. FctB from strain 90/306S was crystallized by the sitting-drop vapour-diffusion method using sodium citrate as a precipitant. The hexagonal FctB crystals belonged to space group P61 or P65, with unit-cell parameters a = b = 95.15, c = 100.25 Å, and diffracted to 2.9 Å resolution.

Plants provide a rich source of lead compounds for a variety of diseases. A novel approach combining phytochemistry and chemotaxis assays was developed and used to identify and study the mechanisms of action of the active compounds in F. japonica, a medicinal herb traditionally used to treat inflammation. Based on a bioactivity-guided purification strategy, two anthranoids, emodin and physcion, were identified from F. japonica. Spectroscopic techniques were used to characterize its crude extract, fractions and phytochemicals. The crude extract, chloroform fraction, and anthranoids of F. japonica significantly inhibited CXCR4-mediated chemotaxis. Mechanistic studies showed that emodin and physcion inhibited chemotaxis via inactivating the MEK/ERK pathway. Moreover, the crude extract and emodin could prevent or treat type 1 diabetes in non-obese diabetic (NOD) mice. This study illustrates the applicability of a combinational approach for the study of anti-inflammatory medicine and shows the potential of F. japonica and its anthranoids for anti-inflammatory therapy.

Background

LNX (Ligand of Numb Protein-X) proteins typically contain an amino-terminal RING domain adjacent to either two or four PDZ domains - a domain architecture that is unique to the LNX family. LNX proteins function as E3 ubiquitin ligases and their domain organisation suggests that their ubiquitin ligase activity may be targeted to specific substrates or subcellular locations by PDZ domain-mediated interactions. Indeed, numerous interaction partners for LNX proteins have been identified, but the in vivo functions of most family members remain largely unclear.

Results

To gain insights into their function we examined the phylogenetic origins and evolution of the LNX gene family. We find that a LNX1/LNX2-like gene arose in an early metazoan lineage by gene duplication and fusion events that combined a RING domain with four PDZ domains. These PDZ domains are closely related to the four carboxy-terminal domains from multiple PDZ domain containing protein-1 (MUPP1). Duplication of the LNX1/LNX2-like gene and subsequent loss of PDZ domains appears to have generated a gene encoding a LNX3/LNX4-like protein, with just two PDZ domains. This protein has novel carboxy-terminal sequences that include a potential modular LNX3 homology domain. The two ancestral LNX genes are present in some, but not all, invertebrate lineages. They were, however, maintained in the vertebrate lineage, with further duplication events giving rise to five LNX family members in most mammals. In addition, we identify novel interactions of LNX1 and LNX2 with three known MUPP1 ligands using yeast two-hybrid asssays. This demonstrates conservation of binding specificity between LNX and MUPP1 PDZ domains.

Conclusions

The LNX gene family has an early metazoan origin with a LNX1/LNX2-like protein likely giving rise to a LNX3/LNX4-like protein through the loss of PDZ domains. The absence of LNX orthologs in some lineages indicates that LNX proteins are not essential in invertebrates. In contrast, the maintenance of both ancestral LNX genes in the vertebrate lineage suggests the acquisition of essential vertebrate specific functions. The revelation that the LNX PDZ domains are phylogenetically related to domains in MUPP1, and have common binding specificities, suggests that LNX and MUPP1 may have similarities in their cellular functions.

Following replication arrest the Cdc25 phosphatase is phosphorylated and inhibited by Cds1. It has previously been reported that expressing Cdc25 where 9 putative amino-terminal Cds1 phosphorylation sites have been substituted to alanine results in bypass of the DNA replication checkpoint. However, these results were acquired by expression of the phosphorylation mutant using a multicopy expression vector in a genetic background where the DNA replication checkpoint is intact. In order to clarify these results we constructed a Cdc25(9A)-GFP native promoter integrant and examined its effect on the replication checkpoint at endogenous expression levels. In this strain the replication checkpoint operates normally, conditional on the presence of the Mik1 kinase. In response to replication arrest the Cdc25(9A)-GFP protein is degraded, suggesting the presence of a backup mechanism to eliminate the phosphatase when it cannot be inhibited through phosphorylation.

Background

Serological tests for IgM and IgG are routinely used in clinical laboratories for the rapid diagnosis of dengue and can differentiate between primary and secondary infections. Dengue virus non-structural protein 1 (NS1) has been identified as an early marker for acute dengue, and is typically present between days 1–9 post-onset of illness but following seroconversion it can be difficult to detect in serum.

Aims

To evaluate the performance of a newly developed Panbio® Dengue Early Rapid test for NS1 and determine if it can improve diagnostic sensitivity when used in combination with a commercial IgM/IgG rapid test.

Methodology

The clinical performance of the Dengue Early Rapid was evaluated in a retrospective study in Vietnam with 198 acute laboratory-confirmed positive and 100 negative samples. The performance of the Dengue Early Rapid in combination with the IgM/IgG Rapid test was also evaluated in Malaysia with 263 laboratory-confirmed positive and 30 negative samples.

Key Results

In Vietnam the sensitivity and specificity of the test was 69.2% (95% CI: 62.8% to 75.6%) and 96% (95% CI: 92.2% to 99.8) respectively. In Malaysia the performance was similar with 68.9% sensitivity (95% CI: 61.8% to 76.1%) and 96.7% specificity (95% CI: 82.8% to 99.9%) compared to RT-PCR. Importantly, when the Dengue Early Rapid test was used in combination with the IgM/IgG test the sensitivity increased to 93.0%. When the two tests were compared at each day post-onset of illness there was clear differentiation between the antigen and antibody markers.

Conclusions

This study highlights that using dengue NS1 antigen detection in combination with anti-glycoprotein E IgM and IgG serology can significantly increase the sensitivity of acute dengue diagnosis and extends the possible window of detection to include very early acute samples and enhances the clinical utility of rapid immunochromatographic testing for dengue.

Author Summary

Dengue is a serious public health concern with around 3 billion people at risk of infection. Severe forms of the infection can be fatal and with no licensed vaccine or effective therapeutic currently available, early detection is important to assist with the clinical management of symptoms. Isolation of the virus and the detection of viral RNA using RT-PCR are commonly used methods for early diagnosis but are time-consuming, expensive and require skilled operation. Rapid immunochromatographic tests (ICT) are relatively simple, inexpensive and easy to perform at or near the point of care. Here, we report on the clinical performance of a new rapid ICT for the non-structural protein 1 (NS1) of dengue virus, a marker of acute infection. At two clinical study sites, NS1 was detected in 60–70% of laboratory-confirmed dengue cases and specificity of the test was >95%. We have also shown that a combined testing approach for both circulating NS1 antigen and antibody responses to the glycoprotein E of the virus can significantly improve diagnostic sensitivity compared to the detection of NS1 alone. Importantly, the combined antigen and antibody testing approach also provides an expanded window of detection from as early as day 1 post-onset of illness.

To facilitate functional analysis of neuronal connectivity in a mammalian nervous system tightly packed with billions of cells, we developed a new technique that allows inducible genetic manipulations within fluorescently labeled single neurons in mice. We term this technique SLICK for Single-neuron Labeling with Inducible Cre-mediated Knockout. SLICK is achieved by co-expressing a drug-inducible form of cre recombinase and a fluorescent protein within the same small subsets of neurons. Thus, SLICK combines the powerful cre recombinase system for conditional genetic manipulation and the fluorescent labeling of single neurons for imaging. We demonstrate efficient inducible genetic manipulation in several types of neurons using SLICK. Furthermore, we apply SLICK to eliminate synaptic transmission in a small subset of neuromuscular junctions. Our results provide evidence for the long-term stability of inactive neuromuscular synapses in adult animals. More broadly, these studies demonstrate a cre-LoxP compatible system for dissecting gene functions in single identifiable neurons.

We describe the magnitude and kinetics of plasma viremia and nonstructural protein 1 (sNS1) levels in sequential samples from 167 children with acute dengue, enrolled early in a community study in Vietnam. All children recovered fully, and only 5 required hospitalization. Among those with dengue virus type 1 (DENV-1), plasma viremia was significantly greater in primary (49) than secondary (44) infections and took longer to resolve. In primary DENV-2 and 3 infections, viremia was significantly lower than among primary DENV-1 infections. Concentrations of sNS1 were significantly higher for DENV-1 than for DENV-2 after adjusting for viremia, with marked differences in the kinetic profiles between primary and secondary infections. Secondary infection and higher viremia were independent predictors of more severe thrombocytopenia, and higher viremia was associated with a small increase in hemoconcentration. Our findings identify clear serotype and immune-status related effects on the dynamics of dengue viremia and sNS1 responses, together with associations with important clinical parameters.

This study was carried out with fresh Australian lager beer which was sampled directly off the production line, the same samples aged for 12 weeks at 30 °C, and the vintage beer which was kept at 20 °C for 5 years. Characteristic Australian lager flavour was maintained in the fresh and vintage beers but was lost in the aged beer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and free thiol group labelling analyses of beer proteins found that this flavour stability correlated with the presence of an unknown 10 kilodaltons (kDa) protein with a higher level of free thiols. The protein was purified by size-exclusion chromatography, then peptide sequencing and database matching identified it as the barley lipid transfer protein (LTP1). Further characterisation using diphenylpicrylhydrazyl (DPPH) free radical scavenging and a Saccharomyces cerevisiae-based antioxidant screening assay demonstrated that the LTP1 protein was active in DPPH reduction and antioxidant activity. The absence of free thiol in the aged beer indicates that the thiol functional groups within the LTP1 protein were saturated and suggests that it is important in the flavour stability of beer by maintaining reduction capacity during the ageing process.

A solid lipid microparticle system containing budesonide was prepared by oil in water emulsification followed by spray drying. The solid lipid system was studied in terms of morphology, particle size distribution, crystallinity, thermal properties, aerosol performance, and dissolution/diffusion release. The microparticle system was also compared to conventional spray-dried crystalline and amorphous budesonide samples. The particle size distributions of the crystalline, amorphous, and solid lipid microparticles, measured by laser diffraction, were similar; however, the microparticle morphology was more irregular than the spray-dried drug samples. The thermal response of the solid lipid microparticles suggested polymorphic transition and melting of the lipid, glycerol behenate (at ~48°C and ~72°C). No budesonide melting or crystallisation peaks were observed, suggesting that the budesonide was integrated into the matrix. X-ray powder diffraction patterns of the crystalline and amorphous budesonide were consistent with previous studies while the solid lipid microparticles showed two peaks, at approximately 21.3 and 23.5 2θ suggesting the metastable sub-α and primarily β′ form. Analysis of the in vitro diffusion/dissolution of the formulations was studied using a flow through model and curves analysed using difference/similarity factors and fitted using the Higuchi model. Regression analysis of this data set indicated differences in the t0.5, where values of 49.7, 35.3, and 136.9 min were observed for crystalline, amorphous, and the solid lipid microparticles, respectively. The aerosol performance (<5 μm), measured by multistage liquid impinger, was 29.5%, 27.3%, and 21.1 ± 0.6% for the crystalline, amorphous, and the solid lipid microparticles, respectively. This study has shown that solid lipid microparticles may provide a useful approach to controlled release respiratory therapy.

Background

Dengue is currently the most important arthropod-borne viral disease of humans. Recent work has shown dengue virus displays limited replication in its primary vector, the mosquito Aedes aegypti, when the insect harbors the endosymbiotic bacterium Wolbachia pipientis. Wolbachia-mediated inhibition of virus replication may lead to novel methods of arboviral control, yet the functional and cellular mechanisms that underpin it are unknown.

Methodology/Principal Findings

Using paired Wolbachia-infected and uninfected Aedes-derived cell lines and dengue virus, we confirm the phenomenon of viral inhibition at the cellular level. Although Wolbachia imposes a fitness cost to cells via reduced proliferation, it also provides a significant degree of protection from virus-induced mortality. The extent of viral inhibition is related to the density of Wolbachia per cell, with highly infected cell lines showing almost complete protection from dengue infection and dramatically reduced virus titers compared to lines not infected with the bacteria.

Conclusions/Significance

We have shown that cells infected with Wolbachia display inhibition of dengue virus replication, that the extent of inhibition is related to bacterial density and that Wolbachia infection, although costly, will provide a fitness benefit in some circumstances. Our results parallel findings in mosquitoes and flies, indicating that cell line models will provide useful and experimentally tractable models to study the mechanisms underlying Wolbachia-mediated protection from viruses.

Edge Hill virus (EHV) is a mosquito-borne flavivirus isolated throughout Australia during mosquito surveillance programs. While not posing an immediate threat to the human population, EHV is a taxonomically interesting flavivirus since it remains the only member of the yellow fever virus (YFV) sub-group to be detected within Australia. Here we present both an antigenic and genetic investigation of collected isolates, and confirm taxonomic classification of the virus within the YFV-group. Isolates were not clustered based on geographical origin or time of isolation, suggesting that minimal genetic evolution of EHV has occurred over geographic distance or time within the EHV cluster. However, two isolates showed significant differences in antigenic reactivity patterns, and had a much larger divergence from the EHV prototype (19% nucleotide and 6% amino acid divergence), indicating a distinct subtype or variant within the EHV subgroup.

The common pathogen Streptococcus pyogenes colonizes the human skin and tonsils and can invade underlying tissues. This requires the adhesion of S. pyogenes to host surface receptors mediated through adhesins. The laminin-binding protein Lbp has been suggested as an adhesin, specific for the human extracellular matrix protein laminin. Sequence alignments, however, indicate a relationship between Lbp and a family of bacterial metal-binding receptors. To further analyze the role of Lbp in S. pyogenes and its potential role in pathogenicity, Lbp has been crystallized, and its structure has been solved at a resolution of 2.45 Å (R = 0.186; Rfree = 0.251). Lbp has the typical metal-binding receptor fold, comprising two globular (β/α)4 domains connected by a helical backbone. The two domains enclose the metal-binding site, which contains a zinc ion. The interaction of Lbp with laminin was further investigated and shown to be specific in vitro. Localization studies with antibodies specific for Lbp show that the protein is attached to the membrane. The data suggest that Lbp is primarily a zinc-binding protein, and we suggest that its interaction with laminin in vivo may be mediated via zinc bound to laminin.

Bladder cancer is the fifth most commonly diagnosed malignancy in the United States and one of the most prevalent worldwide. It harbors a probability of recurrence of >50%, thus rigorous, long-term surveillance of patients is advocated. Flexible cystoscopy coupled with voided urine cytology (VUC) is the primary diagnostic approach, but cystoscopy is an uncomfortable, invasive procedure and the sensitivity of VUC is poor in all but high-grade tumors. Thus, improvements in non-invasive urinalysis assessment strategies would benefit patients. We applied gene expression microarray analysis to exfoliated urothelia recovered from bladder washes obtained prospectively from 46 patients with subsequently confirmed presence or absence of bladder cancer. Data from microarrays containing 56,000 targets was subjected to a panel of statistical analyses to identify bladder cancer-associated gene signatures. Hierarchical clustering and supervised learning algorithms were used to classify samples on the basis of tumor burden. A differentially expressed geneset of 319 gene probes was associated with the presence of bladder cancer (P<0.01), and visualization of protein interaction networks revealed VEGF and AGT as pivotal factors in tumor cells. Supervised machine learning and a cross-validation approach were used to build a 14-gene molecular classifier that was able to classify patients with and without bladder cancer with an overall accuracy of 76%. Our results show that it is possible to achieve the detection of bladder cancer using molecular signatures present in exfoliated tumor urothelia. Further investigation and validation of the cancer-associated profiles may reveal important biomarkers for the non-invasive detection and surveillance of bladder cancer.

Aminoglycoside-2′′-phosphotransferase-IIa [APH(2′′)-IIa] is one of a number of homologous bacterial enzymes responsible for the deactivation of the aminoglycoside family of antibiotics and is thus a major component in bacterial resistance to these compounds. APH(2′′)-IIa produces resistance to several clinically important aminoglycosides (including kanamycin and gentamicin) in both gram-positive and gram-negative bacteria, most notably in Enterococcus species. We have determined the structures of two complexes of APH(2′′)-IIa, the binary gentamicin complex and a ternary complex containing adenosine-5′-(β,γ-methylene)triphosphate (AMPPCP) and streptomycin. This is the first crystal structure of a member of the APH(2′′) family of aminoglycoside phosphotransferases. The structure of the gentamicin-APH(2′′)-IIa complex was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and was refined to a crystallographic R factor of 0.210 (Rfree, 0.271) at a resolution of 2.5 Å. The structure of the AMPPCP-streptomycin complex was solved by molecular replacement using the gentamicin-APH(2′′)-IIa complex as the starting model. The enzyme has a two-domain structure with the substrate binding site located in a cleft in the C-terminal domain. Gentamicin binding is facilitated by a number of conserved acidic residues lining the binding cleft, with the A and B rings of the substrate forming the majority of the interactions. The inhibitor streptomycin, although binding in the same pocket as gentamicin, is orientated such that no potential phosphorylation sites are adjacent to the catalytic aspartate residue. The binding of gentamicin and streptomycin provides structural insights into the substrate selectivity of the APH(2′′) subfamily of aminoglycoside phosphotransferases, specifically, the selectivity between the 4,6-disubstituted and the 4,5-disubstituted aminoglycosides.

Surface energy data for samples of microcrystalline cellulose have been obtained using two techniques: capillary intrusion and inverse gas chromatography. Ten microcrystalline cellulose materials, studied using capillary intrusion, showed significant differences in the measured surface energetics (in terms of total surface energy and the acid–base characteristics of the cellulose surface), with variations noted between the seven different manufacturers who produced the microcrystalline cellulose samples. The surface energy data from capillary intrusion was similar to data obtained using inverse gas chromatography with the column maintained at 44% relative humidity for the three samples of microcrystalline cellulose studied. This suggests that capillary intrusion may be a suitable method to study the surface energy of pharmaceutical samples.

The aim of this research was to determine the reference ultrasonic velocity (v) and attenuation coefficient (α) for 2H, 3H-perfluoropentane (HPFP), 1,1,1,2-tetrafluoroethane (HFA-134a) and 1,1,1,2,3,3,3-tetrafluoroethane (HFA-227) propellants, for the future purpose of characterising pressurised metered dose inhaler (pMDI) formulations using high-resolution ultrasonic spectroscopy (HRUS). Perfluoroheptane (PFH) was used as a reference material for HPFP. With its velocity and attenuation coefficient determined at 25 °C, HPFP was subsequently used as a reference for HFA-134a and HFA-227. It was found that there is a linear decline in ultrasonic velocity with an increase in temperature. As with HPFP, the ultrasonic velocity of HFA-134a and HFA-227 were successfully calculated at 25 °C. However, the difference in density and viscosity between reference and sample prevented accurate determination of reference attenuation coefficient for the hydrofluoroalkanes. With ultrasonic velocity alone, dispersion concentration and stability monitoring for experimental pMDI formulations is possible using HRUS. However, at this point in time measurement of particle size is not feasible.