Ureaplasma parvum : understanding the complexities of intra-amniotic infection in an ovine model

The human Ureaplasma species are the most frequently isolated bacteria from the upper genital tract of pregnant women and can cause clinically asymptomatic, intra-uterine infections, which are difficult to treat with antimicrobials. Ureaplasma infection of the upper genital tract during pregnancy has been associated with numerous adverse outcomes including preterm birth, chorioamnionitis and neonatal respiratory diseases. The mechanisms by which ureaplasmas are able to chronically colonise the amniotic fluid and avoid eradication by (i) the host immune response and (ii) maternally-administered antimicrobials, remain virtually unexplored. To address this gap within the literature, this study investigated potential mechanisms by which ureaplasmas are able to cause chronic, intra-amniotic infections in an established ovine model. In this PhD program of research the effectiveness of standard, maternal erythromycin for the treatment of chronic, intra-amniotic ureaplasma infections was evaluated. At 55 days of gestation pregnant ewes received an intra-amniotic injection of either: a clinical Ureaplasma parvum serovar 3 isolate that was sensitive to macrolide antibiotics (n = 16); or 10B medium (n = 16). At 100 days of gestation, ewes were then randomised to receive either maternal erythromycin treatment (30 mg/kg/day for four days) or no treatment. Ureaplasmas were isolated from amniotic fluid, chorioamnion, umbilical cord and fetal lung specimens, which were collected at the time of preterm delivery of the fetus (125 days of gestation). Surprisingly, the numbers of ureaplasmas colonising the amniotic fluid and fetal tissues were not different between experimentally-infected animals that received erythromycin treatment or infected animals that did not receive treatment (p > 0.05), nor were there any differences in fetal inflammation and histological chorioamnionitis between these groups (p > 0.05). These data demonstrate the inability of maternal erythromycin to eradicate intra-uterine ureaplasma infections. Erythromycin was detected in the amniotic fluid of animals that received antimicrobial treatment (but not in those that did not receive treatment) by liquid chromatography-mass spectrometry; however, the concentrations were below therapeutic levels (<10 – 76 ng/mL). These findings indicate that the ineffectiveness of standard, maternal erythromycin treatment of intra-amniotic ureaplasma infections may be due to the poor placental transfer of this drug. Subsequently, the phenotypic and genotypic characteristics of ureaplasmas isolated from the amniotic fluid and chorioamnion of pregnant sheep after chronic, intra-amniotic infection and low-level exposure to erythromycin were investigated. At 55 days of gestation twelve pregnant ewes received an intra-amniotic injection of a clinical U. parvum serovar 3 isolate, which was sensitive to macrolide antibiotics. At 100 days of gestation, ewes received standard maternal erythromycin treatment (30 mg/kg/day for four days, n = 6) or saline (n = 6). Preterm fetuses were surgically delivered at 125 days of gestation and ureaplasmas were cultured from the amniotic fluid and the chorioamnion. The minimum inhibitory concentrations (MICs) of erythromycin, azithromycin and roxithromycin were determined for cultured ureaplasma isolates, and antimicrobial susceptibilities were different between ureaplasmas isolated from the amniotic fluid (MIC range = 0.08 – 1.0 mg/L) and chorioamnion (MIC range = 0.06 – 5.33 mg/L). However, the increased resistance to macrolide antibiotics observed in chorioamnion ureaplasma isolates occurred independently of exposure to erythromycin in vivo. Remarkably, domain V of the 23S ribosomal RNA gene (which is the target site of macrolide antimicrobials) of chorioamnion ureaplasmas demonstrated significant variability (125 polymorphisms out of 422 sequenced nucleotides, 29.6%) when compared to the amniotic fluid ureaplasma isolates and the inoculum strain. This sequence variability did not occur as a consequence of exposure to erythromycin, as the nucleotide substitutions were identical between chorioamnion ureaplasmas isolated from different animals, including those that did not receive erythromycin treatment. We propose that these mosaic-like 23S ribosomal RNA gene sequences may represent gene fragments transferred via horizontal gene transfer. The significant differences observed in (i) susceptibility to macrolide antimicrobials and (ii) 23S ribosomal RNA sequences of ureaplasmas isolated from the amniotic fluid and chorioamnion suggests that the anatomical site from which they were isolated may exert selective pressures that alter the socio-microbiological structure of the bacterial population, by selecting for genetic changes and altered antimicrobial susceptibility profiles. The final experiment for this PhD examined antigenic size variation of the multiple banded antigen (MBA, a surface-exposed lipoprotein and predicted ureaplasmal virulence factor) in chronic, intra-amniotic ureaplasma infections. Previously defined ‘virulent-derived’ and ‘avirulent-derived’ clonal U. parvum serovar 6 isolates (each expressing a single MBA protein) were injected into the amniotic fluid of pregnant ewes (n = 20) at 55 days of gestation, and amniotic fluid was collected by amniocentesis every two weeks until the time of near-term delivery of the fetus (at 140 days of gestation). Both the avirulent and virulent clonal ureaplasma strains generated MBA size variants (ranging in size from 32 – 170 kDa) within the amniotic fluid of pregnant ewes. The mean number of MBA size variants produced within the amniotic fluid was not different between the virulent (mean = 4.2 MBA variants) and avirulent (mean = 4.6 MBA variants) ureaplasma strains (p = 0.87). Intra-amniotic infection with the virulent strain was significantly associated with the presence of meconium-stained amniotic fluid (p = 0.01), which is an indicator of fetal distress in utero. However, the severity of histological chorioamnionitis was not different between the avirulent and virulent groups. We demonstrated that ureaplasmas were able to persist within the amniotic fluid of pregnant sheep for 85 days, despite the host mounting an innate and adaptive immune response. Pro-inflammatory cytokines (interleukin (IL)-1â, IL-6 and IL-8) were elevated within the chorioamnion tissue of pregnant sheep from both the avirulent and virulent treatment groups, and this was significantly associated with the production of anti-ureaplasma IgG antibodies within maternal sera (p < 0.05). These findings suggested that the inability of the host immune response to eradicate ureaplasmas from the amniotic cavity may be due to continual size variation of MBA surface-exposed epitopes. Taken together, these data confirm that ureaplasmas are able to cause long-term in utero infections in a sheep model, despite standard antimicrobial treatment and the development of a host immune response. The overall findings of this PhD project suggest that ureaplasmas are able to cause chronic, intra-amniotic infections due to (i) the limited placental transfer of erythromycin, which prevents the accumulation of therapeutic concentrations within the amniotic fluid; (ii) the ability of ureaplasmas to undergo rapid selection and genetic variation in vivo, resulting in ureaplasma isolates with variable MICs to macrolide antimicrobials colonising the amniotic fluid and chorioamnion; and (iii) antigenic size variation of the MBA, which may prevent eradication of ureaplasmas by the host immune response and account for differences in neonatal outcomes. The outcomes of this program of study have improved our understanding of the biology and pathogenesis of this highly adapted microorganism.

Two SusD-like proteins encoded within a polysaccharide utilisation locus of an uncultured ruminant bacteroidetes bind strongly to cellulose

We demonstrate that two characteristic Sus-like proteins encoded within a Polysaccharide Utilisation Locus (PUL) bind strongly to cellulosic substrates and interact with plant primary cell walls. This shows associations between uncultured Bacteroidetes-affiliated lineages and cellulose in the rumen, and thus presents new PUL-derived targets to pursue regarding plant biomass degradation.

Challenges to environmental toxicology and epidemiology : where do we stand and which way do we go?

Modern toxicology investigates a wide array of both old and new health hazards. Priority setting is needed to select agents for research from the plethora of exposure circumstances. The changing societies and a growing fraction of the aged have to be taken into consideration. A precise exposure assessment is of importance for risk estimation and regulation. Toxicology contributes to the exploration of pathomechanisms to specify the exposure metrics for risk estimation. Combined effects of co-existing agents are not yet sufficiently understood. Animal experiments allow a separate administration of agents which can not be disentangled by epidemiological means, but their value is limited for low exposure levels in many of today’s settings. As an experimental science, toxicology has to keep pace with the rapidly growing knowledge about the language of the genome and the changing paradigms in cancer development. During the pioneer era of assembling a working draft of the human genome, toxicogenomics has been developed. Gene and pathway complexity have to be considered when investigating gene–environment interactions. For a best conduct of studies, modern toxicology needs a close liaison with many other disciplines like epidemiology and bioinformatics.

Chemopreventive effect of PSP through targeting of prostate cancer stem cell-like population

Recent evidence suggested that prostate cancer stem/progenitor cells (CSC) are responsible for cancer initiation as well as disease progression. Unfortunately, conventional therapies are only effective in targeting the more differentiated cancer cells and spare the CSCs. Here, we report that PSP, an active component extracted from the mushroom Turkey tail (also known as Coriolus versicolor), is effective in targeting prostate CSCs. We found that treatment of the prostate cancer cell line PC-3 with PSP led to the down-regulation of CSC markers (CD133 and CD44) in a time and dose-dependent manner. Meanwhile, PSP treatment not only suppressed the ability of PC-3 cells to form prostaspheres under non-adherent culture conditions, but also inhibited their tumorigenicity in vivo, further proving that PSP can suppress prostate CSC properties. To investigate if the anti-CSC effect of PSP may lead to prostate cancer chemoprevention, transgenic mice (TgMAP) that spontaneously develop prostate tumors were orally fed with PSP for 20 weeks. Whereas 100% of the mice that fed with water only developed prostate tumors at the end of experiment, no tumors could be found in any of the mice fed with PSP, suggesting that PSP treatment can completely inhibit prostate tumor formation. Our results not only demonstrated the intriguing anti-CSC effect of PSP, but also revealed, for the first time, the surprising chemopreventive property of oral PSP consumption against prostate cancer.

Identification of a novel prostate cancer susceptibility variant in the KLK3 gene transcript

Abstract Genome-wide association studies (GWAS) have identified more than 30 prostate cancer (PrCa) susceptibility loci. One of these (rs2735839) is located close to a plausible candidate susceptibility gene, KLK3, which encodes prostate-specific antigen (PSA). PSA is widely used as a biomarker for PrCa detection and disease monitoring. To refine the association between PrCa and variants in this region, we used genotyping data from a two-stage GWAS using samples from the UK and Australia, and the Cancer Genetic Markers of Susceptibility (CGEMS) study. Genotypes were imputed for 197 and 312 single nucleotide polymorphisms (SNPs) from HapMap2 and the 1000 Genome Project, respectively. The most significant association with PrCa was with a previously unidentified SNP, rs17632542 (combined P = 3.9 × 10−22). This association was confirmed by direct genotyping in three stages of the UK/Australian GWAS, involving 10,405 cases and 10,681 controls (combined P = 1.9 × 10−34). rs17632542 is also shown to be associated with PSA levels and it is a non-synonymous coding SNP (Ile179Thr) in KLK3. Using molecular dynamic simulation, we showed evidence that this variant has the potential to introduce alterations in the protein or affect RNA splicing. We propose that rs17632542 may directly influence PrCa risk.

Identification of vitronectin as a novel insulin-like growth factor-II binding protein

We have previously reported the presence of a 70 kDa insulin-like growth factor (IGF)-II-specific binding protein in chicken serum using Western ligand blotting approaches. In order to ascertain the identity of this 70 kDa IGF-II binding species, the protein has been purified from chicken serum using a combination of ion-exchange and gel-permeation chromatography. Interestingly, amino acid sequencing of the purified protein revealed that it has the same N-terminal sequence as chicken vitronectin (VN). The protein has the ability to specifically bind IGF-II and not IGF-I as determined by ligand blotting, cross-linking and competitive binding assay approaches. In addition, the protein binds 125I-des(l-6)-IGF-II, suggesting that the interaction with IGF-II is different to those with other characterized IGF-binding proteins. Importantly, we have ascertained that both human and bovine VN also specifically bind IGF-II. These results are particularly relevant in the light of the recent report that the urokinase-type plasminogen activator receptor, a protein that also binds VN, has been shown to associate with the cation-independent mannose-6-phosphate/GF-II receptor and suggest a possible role for IGF-II in cell adhesion and invasion.

Like our page : using Facebook to support first year students in their transition to higher education

Facebook is approaching ubiquity in the social habits and practice of many students. However, its use in higher education has been criticised (Maranto & Barton, 2010) because it can remove or blur academic boundaries. Despite these concerns, there is strong potential to use Facebook to support new students to communicate and interact with each other (Cheung, Chiu, & Lee, 2010). This paper shows how Facebook can be used by teaching staff to communicate more effectively with students. Further, it shows how it can provide a way to represent and include beginning students’ thoughts, opinions and feedback as an element of the learning design and responsive feed-forward into lectures and tutorial activities. We demonstrate how an embedded social media strategy can be used to complement and enhance the first year curriculum experience by functioning as a transition device for student support and activating Kift’s (2009) organising principles for first year curriculum design.

Phenotypic Characterization of Prostate Cancer LNCaP Cells Cultured within a Bioengineered Microenvironment

Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D) cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D) cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG) hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD) motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP) cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR) nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to androgen stimulation between the 2D and 3D environments. Therefore, we suggest that the presented 3D culture system represents a powerful tool for high throughput prostate cancer drug testing that recapitulates tumor microenvironment. © 2012 Sieh et al.

Copper-containing mesoporous bioactive glass scaffolds with multifunctional properties of angiogenesis capacity, osteostimulation and antibacterial activity

It is of great importance to develop multifunctional bioactive scaffolds, which combine angiogenesis capacity, osteostimulation, and antibacterial properties for regenerating lost bone tissues. In order to achieve this aim, we prepared copper (Cu)-containing mesoporous bioactive glass (Cu-MBG) scaffolds with interconnective large pores (several hundred micrometer) and well-ordered mesopore channels (around 5 nm). Both Cu-MBG scaffolds and their ionic extracts could stimulate hypoxia-inducible factor (HIF)-1a and vascular endothelial growth factor(VEGF) expression in human bone marrow stromal cells(hBMSCs). In addition, both Cu-MBG scaffolds and their ionic extracts significantly promoted the osteogenic differentiation of hBMSCs by improving their bone-related gene expression (alkaline phosphatase (ALP), osteopontin(OPN) and osteocalcin (OCN)). Furthermore, Cu-MBG scaffolds could maintain a sustained release of ibuprofen and significantly inhibited the viability of bacteria. This study indicates that the incorporation of Cu2þ ions into MBG scaffolds significantly enhances hypoxia-like tissue reaction leading to the coupling of angiogenesis and osteogenesis. Cu2þ ions play an important role to offer the multifunctional properties of MBG scaffold system. This study has demonstrated that it is possible to develop multifunctional scaffolds by combining enhanced angiogenesis potential, osteostimulation, and antibacterial properties for the treatment of large bone defects.