Novel porous scaffolds for tissue engineering cartilage

Damage to cartilage, caused either by disease or injury, affects a large number of people worldwide, severely reducing the patient's quality of life and generating a huge burden on healthcare systems. The limited success of treatment options such as tissue grafts has been the driving force behind much research into tissue engineering strategies for cartilage repair. One of the challenges associated with tissue engineering cartilage is that of generating constructs of clinically relevant sizes since the formation of a crust of tissue at the scaffold periphery restricts the supply of nutrients to the growing tissue. The hypothesis of this thesis was that a tissue engineering system incorporating scaffolds containing both random and anisotropic porosity and a novel flow perfusion bioreactor system would facilitate in vitro tissue formation by enhancing the supply of nutrients to the growing construct. This hypothesis was examined using cartilage as a model tissue. It was shown that scaffolds combining both random and anisotropic porosity (sparse knit scaffolds) had improved flow properties compared to scaffolds containing random porosity alone (needled felt scaffolds). Following studies to characterise the scaffolds and to determine the appropriate conditions for seeding cells into the scaffolds, cartilage formation within the different scaffolds was assessed over a four week culture period. It was found that the flow perfusion system was not as favourable for in vitro cartilage formation as either the commercially available Rotary Cell Culture System (RCCS) or static culture. One of the sparse knit scaffolds (sparse knit 4) and the needled felt were further compared for cartilage formation over an eight week culture period, using static and RCCS culture. With respect to collagen and glycosaminoglycan (GAG) production, cartilage constructs generated from the two scaffold systems were similar. Following static culture it was found that more viable cells were present at the centre of sparse knit 4 scaffolds than needled felt scaffolds. It was therefore concluded that scaffolds combining random and anisotropic porosity were advantageous for culturing tissues in environments where nutrient supply was reliant on diffusion alone

Novel porous scaffolds for tissue engineering cartilage

Damage to cartilage, caused either by disease or injury, affects a large number of people worldwide, severely reducing the patient's quality of life and generating a huge burden on healthcare systems. The limited success of treatment options such as tissue grafts has been the driving force behind much research into tissue engineering strategies for cartilage repair. One of the challenges associated with tissue engineering cartilage is that of generating constructs of clinically relevant sizes since the formation of a crust of tissue at the scaffold periphery restricts the supply of nutrients to the growing tissue. The hypothesis of this thesis was that a tissue engineering system incorporating scaffolds containing both random and anisotropic porosity and a novel flow perfusion bioreactor system would facilitate in vitro tissue formation by enhancing the supply of nutrients to the growing construct. This hypothesis was examined using cartilage as a model tissue. It was shown that scaffolds combining both random and anisotropic porosity (sparse knit scaffolds) had improved flow properties compared to scaffolds containing random porosity alone (needled felt scaffolds). Following studies to characterise the scaffolds and to determine the appropriate conditions for seeding cells into the scaffolds, cartilage formation within the different scaffolds was assessed over a four week culture period. It was found that the flow perfusion system was not as favourable for in vitro cartilage formation as either the commercially available Rotary Cell Culture System (RCCS) or static culture. One of the sparse knit scaffolds (sparse knit 4) and the needled felt were further compared for cartilage formation over an eight week culture period, using static and RCCS culture. With respect to collagen and glycosaminoglycan (GAG) production, cartilage constructs generated from the two scaffold systems were similar. Following static culture it was found that more viable cells were present at the centre of sparse knit 4 scaffolds than needled felt scaffolds. It was therefore concluded that scaffolds combining random and anisotropic porosity were advantageous for culturing tissues in environments where nutrient supply was reliant on diffusion alone

Identification, characterisation and quantification of proteins used in chemical communication

Most animals have excretory systems to remove soluble waste. In humans soluble waste is mainly excreted through the urinary system. Kidneys, urinary bladder and urethra make up this system and are responsible for the production of urine by filtration, reabsorption and secretion. Under normal circumstances urine contains water, creatinine, urea and salts. In humans, the presence of elevated levels of protein or glucose is indicative of medical conditions such as impaired kidney function and diabetes. Some animals are an exception to this. Rodents such as the house mouse (Mus musculus), Norway rat (Rattus norvegicus), bank vole (Myodes glareosin) and Roborovski hamster (Phodopus roborovskii) excrete substantial amounts of protein in their urine yet their renal function remains intact. These proteins belong to the lipocalin family and play an essential part in chemical signalling. Their size (18-19kDa) allows them to escape from being filtered out of the urine during the ultrafiltration step resulting in their excretion in the urine. Many of these proteins share a high sequence identity and genomic data is often incomplete or absent. One aspect of this thesis looks at developing a quantification method for a set of highly homologous lipocalins in mice. Another was to characterise and identify proteins excreted in the harvest mouse (Micromys minutus) and mouse lemur (Microcebus) in the absence of genomic data and see if they are related to the lipocalin family or if they belong to a completely different group of proteins. Using mass spectrometric techniques a method to quantify major urinary proteins (MUPS), lipocalins found in mice, was developed and implemented. A quantification concatemer (QconCAT) was designed to do this and was based on genomic data from the laboratory strain of mouse C57BL/6. MUP isoforms were successfully quantified in both male and female C57BL/6 mice. The QconCAT strategy was also used to assess MUP production during the estrous cycle in female mice. Females express more MUP during the estrous stage with a decline in expression seen during the proestrous. For the second part of this thesis, lipocalin expression in the harvest mouse (Micromys minutus) was investigated. Urine samples collected from male and female harvest mice revealed proteins approximately 18-19 kDa expressed in both sexes. The concentration of protein in urine was much lower than that observed in other rodents. Alternative areas of protein excretion were explored and revealed the same protein to be excreted in much higher concentrations from the saliva and/or paws. Again mass spectrometry was employed to identify and characterise these proteins. A preliminary discovery analysis identified proteins that shared high homology with other lipocalins including MUPS and odorant binding proteins. Intact mass analysis also confirmed the presence of three abundant proteins in both males and females. Anion exchange chromatography was used to separate the proteins for de novo sequence analysis which confirmed that harvest mice excrete proteins belonging to the lipocalin family. The final section of this thesis examines characterising protein expression in the mouse lemur (Microcebus). Although they are classed as primates not rodents, mouse lemurs are known to respond to urinary chemosignals from their conspecifics. Urine samples were collected from two species of mouse lemur - Microcebus murinus and Microcebus lehilahytsara. As mouse lemurs have a specific breeding season samples were collected both in and out of season. Some of the male mouse lemurs from both species expressed a large amount of protein during reproductive season. No protein was observed in females. Intact mass analysis identified a protein at 9388 Da in the M. murinus and 9418 Da in the M. lehilahytsara. Unlike many members of the rodent family who excrete large quantities of lipocalins, de novo sequencing confirmed this protein to be a member of the Whey Acidic Protein family (WAPS). WAPS are expressed across many lineages and have a variety of functions including antibacterial and antifungal action, protease inhibition, tumour suppression and anti-inflammatory activity. No protease inhibition by the mouse lemur protein was observed and further studies will need to be established to determine the biological function of this WAP

Glareosin : A novel sexually dimorphic urinary lipocalin in the bank vole, Myodes glareolus

Electronic supplementary material is available online at https://dx.doi.org/10.6084/m9.figshare.c.3859369. Detailed methods are presented in the electronic supplementary material. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [32] partner repository with the dataset identifier PXD006645 and 10.6019/PXD006645 This work was funded in part by BBSRC (BB/J002631/1 and BB/M012557/1).Peer reviewedPublisher PD

Flinders Island spotted fever rickettsioses caused by "marmionii" strain of rickettsia honei, Eastern Australia

Australia has 4 rickettsial diseases: murine typhus, Queensland tick typhus, Flinders Island spotted fever, and scrub typhus. We describe 7 cases of a rickettsiosis with an acute onset and symptoms of fever (100%), headache (71%), arthralgia (43%), myalgia (43%), cough (43%), maculopapular/petechial rash (43%), nausea (29%), pharyngitis (29%), lymphadenopathy (29%), and eschar (29%). Cases were most prevalent in autumn and from eastern Australia, including Queensland, Tasmania, and South Australia. One patient had a history of tick bite (Haemaphysalis novaeguineae). An isolate shared 99.2%, 99.8%, 99.8%, 99.9%, and 100% homology with the 17 kDa, ompA, gltA, 16S rRNA, and Sca4 genes, respectively, of Rickettsia honei. This Australian rickettsiosis has similar symptoms to Flinders Island spotted fever, and the strain is genetically related to R. honei. It has been designated the "marmionii" strain of R. honei, in honor of Australian physician and scientist Barrie Marmion

Population Pharmacokinetics and Pharmacodynamics of Itraconazole for Disseminated Infection Caused by Talaromyces marneffei.

First-line treatment of talaromycosis with amphotericin B deoxycholate (DAmB) is labor-intensive and toxic. Itraconazole is an appealing alternative antifungal agent. Pharmacokinetic data were obtained from 76 patients who were randomized to itraconazole in the Itraconazole versus Amphotericin B for Talaromycosis (IVAP) trial. Plasma levels of itraconazole and its active metabolite, hydroxyitraconazole, were analyzed alongside longitudinal fungal CFU counts in a population model. Itraconazole and hydroxyitraconazole pharmacokinetic variability was considerable, with areas under the concentration-time curve over 24 h (AUC24) of 3.34 ± 4.31 mg·h/liter and 3.57 ± 4.46 mg·h/liter (mean ± standard deviation), respectively. Levels of both analytes were low; itraconazole minimum concentration (Cmin) was 0.11 ± 0.16 mg/liter, and hydroxyitraconazole Cmin was 0.13 ± 0.17 mg/liter. The mean maximal rates of drug-induced killing were 0.206 and 0.208 log10 CFU/ml/h, respectively. There were no associations between itraconazole Cmin/MIC and time to sterilization of the bloodstream (hazard ratio [HR], 1.01; 95% confidence interval [CI], 0.99 to 1.03; P = 0.43), time to death (HR, 0.99; 95% CI, 0.96 to 1.02; P = 0.77), or early fungicidal activity (EFA) (coefficient, -0.004; 95% CI, -0.010 to 0.002; P = 0.18). Similarly, there was no relationship between AUC/MIC and time to sterilization of the bloodstream (HR, 1.00; 95% CI, 0.99 to 1.00; P = 0.50), time to death (HR, 1.00; 95% CI, 0.99 to 1.00; P = 0.91), or EFA (coefficient, -0.0001; 95% CI, -0.0003 to 0.0001; P = 0.19). This study raises the possibility that the failure of itraconazole to satisfy noninferiority criteria against DAmB for talaromycosis in the IVAP trial was a pharmacokinetic and pharmacodynamic failure

The antisaccade task as an index of sustained goal activation in working memory: modulation by nicotine

The antisaccade task provides a laboratory analogue of situations in which execution of the correct behavioural response requires the suppression of a more prepotent or habitual response. Errors (failures to inhibit a reflexive prosaccade towards a sudden onset target) are significantly increased in patients with damage to the dorsolateral prefrontal cortex and patients with schizophrenia. Recent models of antisaccade performance suggest that errors are more likely to occur when the intention to initiate an antisaccade is insufficiently activated within working memory. Nicotine has been shown to enhance specific working memory processes in healthy adults. MATERIALS AND METHODS: We explored the effect of nicotine on antisaccade performance in a large sample (N = 44) of young adult smokers. Minimally abstinent participants attended two test sessions and were asked to smoke one of their own cigarettes between baseline and retest during one session only. RESULTS AND CONCLUSION: Nicotine reduced antisaccade errors and correct antisaccade latencies if delivered before optimum performance levels are achieved, suggesting that nicotine supports the activation of intentions in working memory during task performance. The implications of this research for current theoretical accounts of antisaccade performance, and for interpreting the increased rate of antisaccade errors found in some psychiatric patient groups are discussed

Molecular pharmacodynamics of meropenem for nosocomial pneumonia caused by <i>Pseudomonas aeruginosa</i>.

ImportanceThe emergence of antimicrobial resistance (AMR) during antimicrobial treatment for hospital-acquired pneumonia (HAP) is a well-documented problem (particularly in pneumonia caused by Pseudomonas aeruginosa) that contributes to the wider global antimicrobial resistance crisis. During drug development, regimens are typically determined by their sufficiency to achieve bactericidal effect. Prevention of the emergence of resistance pharmacodynamics is usually not characterized or used to determine the regimen. The innovative experimental platform described here allows characterization of the emergence of AMR during the treatment of HAP and the development of strategies to mitigate this. We have demonstrated this specifically for meropenem-a broad-spectrum antibiotic commonly used to treat HAP. We have characterized the antimicrobial resistance pharmacodynamics of meropenem when used to treat HAP, caused by initially meropenem-susceptible P. aeruginosa, phenotypically and genotypically. We have also shown that intensifying the regimen and using combination therapy are both strategies that can both treat HAP and suppress the emergence of resistance

Latitude, temperature, and habitat complexity predict predation pressure in eelgrass beds across the Northern Hemisphere

Latitudinal gradients in species interactions are widely cited as potential causes or consequences of global patterns of biodiversity. However, mechanistic studies documenting changes in interactions across broad geographic ranges are limited. We surveyed predation intensity on common prey (live amphipods and gastropods) in communities of eelgrass (Zostera marina) at 48 sites across its Northern Hemisphere range, encompassing over 370 of latitude and four continental coastlines. Predation on amphipods declined with latitude on all coasts but declined more strongly along western ocean margins where temperature gradients are steeper. Whereas in situ water temperature at the time of the experiments was uncorrelated with predation, mean annual temperature strongly positively predicted predation, suggesting a more complex mechanism than simple increased metabolic activity at the time of predation. This large-scale biogeographic pattern was modified by local habitat characteristics; predation declined with higher shoot density both among and within sites. Predation rates on gastropods, by contrast, were uniformly low and varied little among sites. The high replication and geographic extent of our study not only provides additional evidence to support biogeographic variation in intensity, but also insight into the mechanisms that relate temperature and biogeographic gradients in species interactions

The biogeography of community assembly: latitude and predation drive variation in community trait distribution in a guild of epifaunal crustaceans

While considerable evidence exists of biogeographic patterns in the intensity of species interactions, the influence of these patterns on variation in community structure is less clear. Studying how the distributions of traits in communities vary along global gradients can inform how variation in interactions and other factors contribute to the process of community assembly. Using a model selection approach on measures of trait dispersion in crustaceans associated with eelgrass (Zostera marina) spanning 30 degrees of latitude in two oceans, we found that dispersion strongly increased with increasing predation and decreasing latitude. Ocean and epiphyte load appeared as secondary predictors; Pacific communities were more overdispersed while Atlantic communities were more clustered, and increasing epiphytes were associated with increased clustering. By examining how species interactions and environmental filters influence community structure across biogeographic regions, we demonstrate how both latitudinal variation in species interactions and historical contingency shape these responses. Community trait distributions have implications for ecosystem stability and functioning, and integrating large-scale observations of environmental filters, species interactions and traits can help us predict how communities may respond to environmental change.info:eu-repo/semantics/publishedVersio