Characterisation of Ilomastat for Prolonged Ocular Drug Release

We are developing tablet dosage forms for implantation directly into the subconjunctival space of the eye. The matrix metalloproteinase inhibitor, ilomastat, has previously been shown to be efficacious at suppressing scarring following glaucoma filtration surgery (GFS). We report on the physical characterisation of ilomastat which is being developed for ocular implantation. Since ilomastat is being considered for implantation it is necessary to examine its polymorphs and their influence on aspects of the in vitro drug release profile. X-ray powder diffraction identified two polymorphs of ilomastat from different commercial batches of the compound. Tablets were prepared from the two different polymorphs. Isothermal perfusion calorimetry was used to show that amorphous content is not increased during tablet formulation. The melting points of the two polymorphs are 188 and 208°C as determined by differential scanning calorimetry. Utilising single crystal X-ray diffraction, the structural conformations and packing arrangements of the different polymorphs were determined. The orthorhombic crystal crystallised as a monohydrate while the second monoclinic crystal form is non-solvated. Ilomastat tablets prepared from the two different solid forms exhibited similar drug release profiles in vitro under conditions mimicking the aqueous composition, volume and flow of the subconjunctival space after GFS. This suggests that a reproducible dose at each time point during release after implantation should be achievable in vivo with ilomastat tablets prepared from the two polymorphs identified.     

The devil in the details of life-history evolution: Instability and reversal of genetic correlations during selection on<Emphasis Type="Italic">Drosophila</Emphasis> development

The evolutionary relationships between three major components of Darwinian fitness, development rate, growth rate and preadult survival, were estimated using a comparison of 55 distinct populations ofDrosophila melanogaster variously selected for age-specific fertility, environmental-stress tolerance and accelerated development. Development rate displayed a strong net negative evolutionary correlation with weight at eclosion across all selection treatments, consistent with the existence of a size-versus-time tradeoff between these characters. However, within the data set, the magnitude of the evolutionary correlation depended upon the particular selection treatments contrasted. A previously proposed tradeoff between preadult viability and growth rate was apparent only under weak selection for juvenile fitness components. Direct selection for rapid development led to sharp reductions in both growth rates and viability. These data add to the mounting results from experimental evolution that illustrate the sensitivity of evolutionary correlations to (i) genotype-by-environment (G X E) interaction, (ii) complex functional-trait interactions, and (iii) character definition. Instability, disappearance and reversal of patterns of genetic covariation often occur over short evolutionary time frames and as the direct product of selection, rather than some stochastic process. We suggest that the functional architecture of fitness is a rapidly evolving matrix with reticulate properties, a matrix that we understand only poorly.     

Chondrocalcinosis is common in the absence of knee involvement

Introduction

We aimed to describe the distribution of radiographic chondrocalcinosis (CC) and to examine whether metacarpophalangeal joint (MCPJ) calcification and CC at other joints occurs in the absence of knee involvement.

Methods

This was a cross-sectional study embedded in the Genetics of Osteoarthritis and Lifestyle study (GOAL). All participants (n = 3,170) had radiographs of the knees, hands, and pelvis. These were scored for radiographic changes of osteoarthritis (OA), for CC at knees, hips, symphysis pubis, and wrists, and for MCPJ calcification. The prevalence of MCPJ calcification and CC overall, at each joint, and in the presence or absence of knee involvement, was calculated.

Results

The knee was the commonest site of CC, followed by wrists, hips, and symphysis pubis. CC was more likely to be bilateral at knees and wrists but unilateral at hips. MCPJ calcification was usually bilateral, and less common than CC at knees, hips, wrists, and symphysis pubis. Unlike that previously reported, CC commonly occurred without any knee involvement; 44.4% of wrist CC, 45.9% of hip CC, 45.5% of symphysis pubis CC, and 31.3% of MCPJ calcification occurred in patients without knee CC. Those with meniscal or hyaline articular cartilage CC had comparable ages (P = 0.21), and neither preferentially associated with fibrocartilage CC at distant joints.

Conclusions

CC visualized on a plain radiograph commonly occurs at other joints in the absence of radiographic knee CC. Therefore, knee radiographs alone are an insufficient screening test for CC. This has significant implications for clinical practice, for epidemiologic and genetic studies of CC, and for the definition of OA patients with coexistent crystal deposition.     

A Retained Secretory Signal Peptide Mediates High Density Lipoprotein (HDL) Assembly and Function of Haptoglobin-related Protein

Haptoglobin-related protein (Hpr) is a component of a minor subspecies of high density lipoproteins (HDL) that function in innate immunity. Here we show that assembly of Hpr into HDL is mediated by its retained N-terminal signal peptide, an unusual feature for a secreted protein and the major difference between Hpr and the soluble acute phase protein haptoglobin (Hp). The 18-amino acid signal peptide is necessary for binding to HDL and interacts directly with the hydrocarbon region of lipids. Utilizing model liposomes, we show that the rate of assembly and steady-state distribution of Hpr in lipid particles is mediated by the physical property of lipid fluidity. Dye release assays reveal that Hpr interacts more rapidly with fluid liposomes. Conversely, steady-state binding assays indicate that more rigid lipid compositions stabilize Hpr association. Lipid association also plays a role in facilitating hemoglobin binding by Hpr. Our data may offer an explanation for the distinct distribution of Hpr among HDL subspecies. Rather than protein-protein interactions mediating localization, direct interaction with phospholipids and sensitivity to lipid fluidity may be sufficient for localization of Hpr and may represent a mechanism of HDL subspeciation.     

Transmitted/Founder Simian Immunodeficiency Virus Envelope Sequences in Vesicular Stomatitis and Semliki Forest Virus Vector Immunized Rhesus Macaques

Identification of transmitted/founder simian immunodeficiency virus (SIV) envelope sequences responsible for infection may prove critical for understanding HIV/SIV mucosal transmission. We used single genome amplification and phylogenetic analyses to characterize transmitted/founder SIVs both in the inoculum and in immunized-infected rhesus monkeys. Single genome amplification of the SIVsmE660 inoculum revealed a maximum diversity of 1.4%. We also noted that the consensus sequence of the challenge stock differed from the vaccine construct in 10 amino acids including 3 changes in the V4 loop. Viral env was prepared from rhesus plasma in 3 groups of 6 immunized with vesicular stomatitis virus (VSV) vectors and boosted with Semliki forest virus (SFV) replicons expressing (a) SIVsmE660 gag-env (b) SIVsmE660 gag-env plus rhesus GM-CSF and (c) control influenza hemagglutinin protein. Macaques were immunized twice with VSV-vectors and once with SFV vector and challenged intrarectally with 4000 TCID₅₀. Single genome amplification characterized the infections of 2 unprotected animals in the gag-env immunized group, both of which had reduced acute plasma viral loads that ended as transient infections indicating partial immune control. Four of 6 rhesus were infected in the gag-env + GM-CSF group which demonstrated that GM-CSF abrogated protection. All 6 animals from the control group were infected having high plasma viral loads. We obtained 246 full-length envelope sequences from SIVsmE660 infected macaques at the peak of infection and determined the number of transmitted/founder variants per animal. Our analysis found that 2 of 2 gag-env vaccinated but infected macaques exhibited single but distinct virus envelope lineages whereas rhesus vaccinated with gag-env-GM-CSF or HA control exhibited both single and multiple env lineages. Because there were only 2 infected animals in the gag-env vaccinated rhesus compared to 10 infected rhesus in the other 2 groups, the significance of finding single env variants in the gag-env vaccinated group could not be established.     

The Role of Climatic Factors in the Expression of an Intrasexual Signal in the Paper Wasp Polistes dominulus

Within a species, variation in the use of sexual signals is observed between different populations. For intersexual traits, differences in the environmental conditions experienced by populations can play an important role in driving variation in male ornaments and female preferences. However, little is known about the factors maintaining variation in intrasexual traits used in competition. In this study, we investigate the role of climatic conditions in maintaining variation in the expression of an intrasexual signal in the paper wasp Polistes dominulus. The results of an experiment in which pupae were housed under different temperature and humidity conditions revealed a strong effect of temperature during pupal development on the expression of the signal. Furthermore, a comparison of survival and body weight between wasps reared at different temperatures indicates that signal expression exhibits phenotypic plasticity in response to developmental temperature. The effect of temperature on signal expression is consistent with patterns of signal expression observed across populations in the wild and suggests that climatic conditions may act to constrain signal expression in some populations but not in others, driving variation in signal use within P. dominulus. Environmental conditions may therefore be important in defining the scope for intrasexual signalling in animal populations and, in doing so, may play a role in maintaining variation in intrasexual traits in the face of sexual selection.     

Geographical variation in ribotype profiles of Escherichia coli isolates from humans,swine, poultry,beef, and dairy cattle in Florida

Waters impacted by fecal pollution can exact high risks to human health and can result in financial losses due to closures of water systems used for recreation and for harvesting seafood. Identifying the sources of fecal pollution in water is paramount in assessing the potential human health risks involved as well as in assessing necessary remedial action. Recently, various researchers have used the ribotyping method to identify sources of bacterial indicators (Escherichia coli and enterococci) in environmental waters. While these studies have identified genotypic differences between human- and animal-derived indicators that are capable of differentiating organisms isolated from humans and various animal hosts, most have focused on organisms collected from a confined geographic area and have not addressed the question of whether these ribotype profiles are watershed specific or if they can be applied universally to organisms from other geographic locations. In this study, E. coli isolates were obtained from humans, beef cattle, dairy cattle, swine, and poultry from locations in northern, central, and southern Florida and were subjected to ribotyping analysis. The intent was to determine (i) if ribotype profiles are capable of discriminating the source of E. coli at the host species level and (ii) if the resulting fingerprints are uniform over an extended geographic area or if they can be applied only to a specific watershed. Our research indicated that, using a single restriction enzyme (HindIII), the ribotyping procedure is not capable of differentiating E. coli isolates from the different animal species sampled in this study. Results indicate, however, that this procedure can still be used effectively to differentiate E. coli as being either human or animal derived when applied to organisms isolated from a large geographic region.