New insights into the altered adhesive and mechanical properties of red blood cells parasitized by Babesia bovis

Sequestration of parasite-infected red blood cells (RBCs) in the microvasculature is an important pathological feature of both bovine babesiosis caused by Babesia bovis and human malaria caused by Plasmodium falciparum. Surprisingly, when compared with malaria, the cellular and molecular mechanisms that underlie this abnormal circulatory behaviour for RBCs infected with B. bovis have been relatively ignored. Here, we present some novel insights into the adhesive and mechanical changes that occur in B. bovis-infected bovine RBCs and compare them with the alterations that occur in human RBCs infected with P. falciparum. After infection with B. bovis, bovine RBCs become rigid and adhere to vascular endothelial cells under conditions of physiologically relevant flow. These alterations are accompanied by the appearance of ridge-like structures on the RBC surface that are analogous, but morphologically and biochemically different, to the knob-like structures on the surface of human RBCs infected with P. falciparum. Importantly, albeit for a limited number of parasite lines examined here, the extent of these cellular and rheological changes appear to be related to parasite virulence. Future investigations to identify the precise molecular composition of ridges and the proteins that mediate adhesion will provide important insight into the pathogenesis of both babesiosis and malaria.

"Like a monkey with a miniature cymbal": Cultural practices of repetition in visual social media

Looping media are recurring components of online content, from gifs to Vine videos, in addition to the conceptual repetition of memes and related practices. This paper analyses practices around looping visual media as examples of vernacular creativity, social media literacies, and internet culture, especially for irreverent and playful purposes. Focusing on the LGBTQ digital cultural context as a pilot study, this research examines multi-platform uses of looping media, including personal narratives through Vine videos and animated gifs on Tumblr. In addition to textual analysis of LGBTQ looping visual social media content, the study will further explore the platform context as part of the experience of looped media. The research will address how these factors may also contribute to practices of irreverence and play, both within the specific case of LGBTQ culture and internet culture more generally.

Plant hosts of the phytoplasmas and rickettsia-like-organisms associated with strawberry lethal yellows and green petal diseases

Candidatus Phytoplasma australiense (Ca. P. australiense) is associated with the plant diseases strawberry lethal yellows (SLY), strawberry green petal (SGP), papaya dieback (PDB), Australian grapevine yellows (AGY) and Phormium yellow leaf (PYL; New Zealand). Strawberry lethal yellows disease is also associated with a rickettsia-like-organism (RLO) or infrequently with the tomato big bud (TBB) phytoplasma, the latter being associated with a wide range of plant diseases throughout Australia. In contrast, the RLO has been identified only in association with SLY disease, and Ca. P. australiense has been detected only in a limited number of plant host species. The aim of this study was to identify plant hosts that are possible reservoirs of Ca. P. australiense and the SLY RLO. Thirty-one plant species from south-east Queensland were observed with disease between 2001 and 2003 and, of these, 18 species tested positive using phytoplasma-specific primers. The RLO was detected in diseased Jacksonia scoparia and Modiola caroliniana samples collected at Stanthorpe. The TBB phytoplasma was detected in 16 different plant species and Ca. P. australiense Australian grapevine yellows strain was detected in six species. The TBB phytoplasma was detected in plants collected at Nambour, Stanthorpe, Warwick and Brisbane. Ca. P. australiense was detected in plants collected at Nambour, Stanthorpe, Gatton and Allora. All four phytoplasmas were detected in diseased Gomphocarpus physocarpus plants collected at Toowoomba, Allora, Nambour and Gatton. These results indicated that the vector(s) of Ca. P. australiense are distributed throughout south-east Queensland and the diversity of phytoplasmas detected in G. physocarpus suggests it is a feeding source for phytoplasma insect vectors or it has a broad susceptibility to a range of phytoplasmas.

Rickettsia-like-organisms and phytoplasmas associated with diseases in Australian strawberries

Strawberry lethal yellows (SLY) disease in Australia is associated with the phytoplasmas Candidatus Phytoplasma australiense and tomato big bud, and a rickettsia-like-organism (RLO). Ca. P. australiense is also associated with strawberry green petal (SGP) disease. This study investigated the strength of the association of the different agents with SLY disease. We also documented the location of SLY or SGP plants, and measured whether they were RLO or phytoplasma positive. Symptomatic strawberry plants collected from south-east Queensland (Australia) between January 2000 and October 2002 were screened by PCR for both phytoplasmas and the RLO. Two previously unreported disease symptoms termed severe fruit distortion (SFD) and strawberry leaves from fruit (SLF) were observed during this study but there was no clear association between these symptoms and phytoplasmas or the RLO. Only two SGP diseased plants were observed and collected, compared with 363 plants with SLY disease symptoms. Of the 363 SLY samples, 117 tested positive for the RLO, 67 tested positive for Ca. P. australiense AGY strain and 11 plants tested positive for Ca. P. australiense PYL variant strain. On runner production farms at Stanthorpe, Queensland the RLO was detected in SLY diseased plants more frequently than for the phytoplasmas. On fruit production farms on the Sunshine Coast, Queensland, Ca. P. australiense was detected in SLY disease plants more frequently than the RLO.

The acceptability, usability and short-term outcomes of Get Real: A web-based program for psychotic-like experiences (PLEs)

Background Psychotic-like experiences (PLEs) are subclinical delusional ideas and perceptual disturbances that have been associated with a range of adverse mental health outcomes. This study reports a qualitative and quantitative analysis of the acceptability, usability and short term outcomes of Get Real, a web program for PLEs in young people. Methods Participants were twelve respondents to an online survey, who reported at least one PLE in the previous 3 months, and were currently distressed. Ratings of the program were collected after participants trialled it for a month. Individual semi-structured interviews then elicited qualitative feedback, which was analyzed using Consensual Qualitative Research (CQR) methodology. PLEs and distress were reassessed at 3 months post-baseline. Results User ratings supported the program's acceptability, usability and perceived utility. Significant reductions in the number, frequency and severity of PLE-related distress were found at 3 months follow-up. The CQR analysis identified four qualitative domains: initial and current understandings of PLEs, responses to the program, and context of its use. Initial understanding involved emotional reactions, avoidance or minimization, limited coping skills and non-psychotic attributions. After using the program, participants saw PLEs as normal and common, had greater self-awareness and understanding of stress, and reported increased capacity to cope and accept experiences. Positive responses to the program focused on its normalization of PLEs, usefulness of its strategies, self-monitoring of mood, and information putting PLEs into perspective. Some respondents wanted more specific and individualized information, thought the program would be more useful for other audiences, or doubted its effectiveness. The program was mostly used in low-stress situations. Conclusions The current study provided initial support for the acceptability, utility and positive short-term outcomes of Get Real. The program now requires efficacy testing in randomized controlled trials.

Subtypes of psychotic-like experiences are differentially associated with suicidal ideation, plans and attempts in young adults

Psychotic-like experiences (PLEs) have been associated with increased risk of suicidality, but it is unclear whether the level of risk varies with different types of PLE. A cross-sectional online survey was completed by 1610 university students. Respondents completed the Community Assessment of Psychic Experiences-15 (CAPE-P15) assessing PLEs on three subscales: Perceptual Abnormalities (PA), Persecutory Ideation (PI) and Bizarre Experiences (BE). Lifetime suicidal ideation, plans and attempts, cannabis, ecstasy and methamphetamine use and family history of mental disorder were also assessed. Multinomal logistic regression was used to examine unique determinants of lifetime suicidality, defined as any history of (i) suicidal ideation or plans and (ii) any attempt, relative to no lifetime history of suicidality. A lifetime history of PA and PI provided significant unique contributions to the prediction of suicide risk, after control for other significant predictors. BE were not associated with any suicide variable demonstrating the variation in risk of suicidality with different types of PLEs. Perceptual abnormalities and persecutory ideation as measured by the CAPE-P15 are the PLEs associated with a higher risk of lifetime suicidality.

Current CAPE-15: A measure of recent psychotic-like experiences and associated distress

Aim Psychotic-like experiences (PLEs) are common in young people and are associated with both distress and adverse outcomes. The Community Assessment of Psychic Experiences-Positive Scale (CAPE-P) provides a 20-item measure of lifetime PLEs. A 15-item revision of this scale was recently published (CAPE-P15). Although the CAPE-P has been used to assess PLEs in the last 12 months, there is no version of the CAPE for assessing more recent PLEs (e.g. 3 months). This study aimed to determine the reliability and validity of the current CAPE-P15 and assess its relationship with current distress. Method A cross-sectional online survey of 489 university students (17–25 years) assessed lifetime and current substance use, current distress, and lifetime and 3-month PLEs on the CAPE-P15. Results Confirmatory factor analysis indicated that the current CAPE-P15 retained the same three-factor structure as the lifetime version consisting of persecutory ideation, bizarre experiences and perceptual abnormalities. The total score of the current version was lower than the lifetime version, but the two were strongly correlated (r = .64). The current version was highly predictive of generalized distress (r = .52) and indices that combined symptom frequency with associated distress did not confer greater predictive power than frequency alone. Conclusion This study provided preliminary data that the current CAPE-P15 provides a valid and reliable measure of current PLEs. The current CAPE-P15 is likely to have substantial practical utility if it is later shown to be sensitive to change, especially in prevention and early intervention for mental disorders in young people.

Reversals of polypeptide chain in globular proteins.

A simple algorithm has been developed to detect β-bends and 'loops'-chain reversals containing five amino acid residues, using only coordinates of Cα-atoms from crystal structure data of globular proteins using the above algorithm. Analysis of bends have showed that the total number of bends in each protein (TB) is linearly related to total number of non-hydrophobic residues in that protein which in turn is related linearly to total number of amino acid residues. Secondly, we found that a large number of consecutive bends occur in each protein which give rise to on an average only three independent residues per turn. Positional preference of amino acid residues in chain reversals is stressed. Consideration of pairs of amino acid residues in positions (i + 1) and (i + 2) of bends seems to provide a more reliable basis for predicting chain reversals in proteins.

Development of a New Zealand database of plant virus and virus-like organisms.

The recent 8th Australasian plant virology workshop in Rotorua, New Zealand, discussed the development of a New Zealand database of plant virus and virus-like organisms. Key points of discussion included: (i) the purpose of such a database; (ii) who would benefit from the information in a database; (iii) the scope of a database and its associated collections; (iv) database information and format; and (v) potential funding of such a database. From the workshop and further research, we conclude that the preservation and verification of specimens within the collections and the development of a New Zealand database of plant virus and virus-like organisms is essential. Such a collection will help to fulfil statutory requirements in New Zealand and assist in fulfilling international obligations under the International Plant Protection Convention. Sustaining such a database will assist New Zealand virologists and statutory bodies to undertake scientifically sound research. Establishing reliable records and an interactive database will help to ensure accurate and timely diagnoses of diseases caused by plant viruses and virus-like organisms. Detection of new incursions and their diagnosis will be further enhanced by the use of such reference collections and their associated database. Connecting and associating this information to similar overseas databases would assist international collaborations and allow access to the latest taxonomic and diagnostic resources. Associated scientists working in the areas of plant breeding, export phytosanitary assurance and in the area of the conservation estate would also benefit from access to verified specimens of plant viruses and virus-like organisms. We conclude that funding of a New Zealand database of virus and virus-like organisms and its associated collections should be based partly on Crown funds, as it is a nationally significant biological resource.

Identification of putative virulence-associated genes of Haemophilus parasuis through suppression subtractive hybridization.

Haemophilus parasuis is the causative agent of Glässer's disease. Up to now 15 serovars of H. parasuis have been identified, with significant differences existing in virulence between serovars. In this study, suppression subtractive hybridization (SSH) was used to identify the genetic difference between Nagasaki (H. parasuis serovar 5 reference strain, highly virulent) and SW114 (H. parasuis serovar 3 reference strain, non-virulent). A total of 191 clones were obtained from the SSH library. Using dot hybridization and PCR, 15 clones were identified containing fragments that were present in the Nagasaki genome while absent in the SW114 genome. Among these 15 fragments, three fragments (ssh1, ssh13, ssh15) encode cell surface-associated components; three fragments (ssh2, ssh5, ssh9) are associated with metabolism and stress response; one fragment (ssh8) is involved in assembly of fimbria and one fragment (ssh6) is a phage phi-105 ORF25-like protein. The remaining seven fragments are hypothetical proteins or unknown. Based on PCR analysis of the 15 serovar reference strains, eight fragments (ssh1, ssh2, ssh3, ssh6, ssh8, ssh10, ssh11 and ssh12) were found in three to five of most virulent serovars (1, 5, 10, 12, 13 and 14), zero to two in three moderately virulent serovars (2, 4 and 15), but absent in the low virulent serovar (8) and non-virulent serovars (3, 6, 7, 9 and 11). In vivo transcription fragments ssh1, ssh2, ssh8 and ssh12 were identified in total RNA samples extracted from experimental infected pig lung by RT-PCR. This study has provided some evidence of genetic differences between H. parasuis strains of different virulence.

Epithelial-mesenchymal transition in oral squamous carcinoma cells

In epithelial-mesenchymal transition (EMT), epithelial cells acquire traits typical for mesenchymal cells, dissociate their cell-cell junctions and gain the ability to migrate. EMT is essential during embryogenesis, but may also mediate cancer progression. Basement membranes are sheets of extracellular matrix that support epithelial cells. They have a major role in maintaining the epithelial phenotype and, in cancer, preventing cell migration, invasion and metastasis. Laminins are the main components of basement membranes and may actively contribute to malignancy. We first evaluated the differences between cell lines obtained from oral squamous cell carcinoma and its recurrence. As the results indicated a change from epithelial to fibroblastoid morphology, E-cadherin to N-cadherin switch, and change in expression of cytokeratins to vimentin intermediate filaments, we concluded that these cells had undergone EMT. We further induced EMT in primary tumour cells to gain knowledge of the effects of transcription factor Snail in this cell model. The E-cadherin repressors responsible for the EMT in these cells were ZEB-1, ZEB-2 and Snail, and ectopic expression of Snail was able to augment the levels of ZEB-1 and ZEB-2. We produced and characterized two monoclonal antibodies that specifically recognized Snail in cell lines and patient samples. By immunohistochemistry, Snail protein was found in mesenchymal tissues during mouse embryonal development, in fibroblastoid cells of healing skin wounds and in fibromatosis and sarcoma specimens. Furthermore, Snail localized to the stroma and borders of tumour cell islands in colon adenocarcinoma, and in laryngeal and cervical squamous cell carcinomas. Immunofluorescence labellings, immunoprecipitations and Northern and Western blots showed that EMT induced a progressive downregulation of laminin-332 and laminin-511 and, on the other hand, an induction of mesenchymal laminin-411. Chromatin immunoprecipitation revealed that Snail could directly bind upstream to the transcription start sites of both laminin α5 and α4 chain genes, thus regulating their expression. The levels of integrin α6β4, a receptor for laminin-332, as well as the hemidesmosomal complex proteins HD1/plectin and BP180 were downregulated in EMT-experienced cells. The expression of Lutheran glycoprotein, a specific receptor for laminin-511, was diminished, whereas the levels of integrins α6β1 and α1β1 and integrin-linked kinase were increased. In quantitative cell adhesion assays, the cells adhered potently to laminin-511 and fibronectin, but only marginally to laminin-411. Western blots and immunoprecipitations indicated that laminin-411 bound to fibronectin and could compromise cell adhesion to fibronectin in a dose-dependent manner. EMT induced a highly migratory and invasive tendency in oral squamous carcinoma cells. Actin-based adhesion and invasion structures, podosomes and invadopodia, were detected in the basal cell membranes of primary tumour and spontaneously transformed cancer cells, respectively. Immunofluorescence labellings showed marked differences in their morphology, as podosomes organized a ring structure with HD1/plectin, αII-spectrin, talin, focal adhesion kinase and pacsin 2 around the core filled with actin, cortactin, vinculin and filamin A. Invadopodia had no division between ring and core and failed to organize the ring proteins, but instead assembled tail-like, narrow actin cables that showed a talin-tensin switch. Time-lapse live-cell imaging indicated that both podosomes and invadopodia were long-lived entities, but the tails of invadopodia vigorously propelled in the cytoplasm and were occasionally released from the cell membrane. Invadopodia could also be externalized outside the cytoplasm, where they still retained the ability to degrade matrix. In 3D confocal imaging combined with in situ gelatin zymography, the podosomes of primary tumour cells were large, cylindrical structures that increased in time, whereas the invadopodia in EMT-driven cells were smaller, but more numerous and degraded the underlying matrix in significantly larger amounts. Fluorescence recovery after photobleaching revealed that the substructures of podosomes were replenished more rapidly with new molecules than those of invadopodia. Overall, our results indicate that EMT has a major effect on the transcription and synthesis of both intra- and extracellular proteins, including laminins and their receptors, and on the structure and dynamics of oral squamous carcinoma cells.

Effects of dairy proteins and calcium on diet-induced obesity in mice

Diet high in dairy products is inversely associated with body mass index, risk of metabolic syndrome and prevalence of type 2 diabetes in several populations. Also a number of intervention studies support the role of increased dairy intake in the prevention and treatment of obesity. Dairy calcium has been suggested to account for the effect of dairy on body weight, but it has been repeatedly shown that the effect of dairy is superior to the effect of supplemental calcium. Dairy proteins are postulated to either enhance the effect of calcium or have an independent effect on body weight, but studies in the area are scarce. The aim of this study was to evaluate the potential of dairy proteins and calcium in the prevention and treatment of diet-induced obesity in C57Bl/6J mice. The effect of dairy proteins and calcium on the liver and adipose tissue was also investigated in order to characterise the potential mechanisms explaining the reduction of risk for metabolic syndrome and type 2 diabetes. A high-calcium diet (1.8%) in combination with dietary whey protein inhibited body weight and fat gain and accelerated body weight and fat loss in high-fat-fed C57Bl/6J mice during long-term studies of 14 to 21 weeks. α-lactalbumin, one of the major whey proteins, was the most effective whey protein fraction showing significantly accelerated weight and fat loss during energy restriction and reduced the amount of visceral fat gain during ad libitum feeding after weight loss. The microarray data suggest sensitisation of insulin signalling in the adipose tissue as a result of a calcium-rich whey protein diet. Lipidomic analysis revealed that weight loss on whey protein-based high-calcium diet was characterised by significant decreases in diabetogenic diacylglycerols and lipotoxic ceramide species. The calcium supplementation led to a small, but statistically significant decrease in fat absorption independent of the protein source of the diet. This augments, but does not fully explain the effects of the studied diets on body weight. A whey protein-containing high-calcium diet had a protective effect against a high-fat diet-induced decline of β3 adrenergic receptor expression in adipose tissue. In addition, a high-calcium diet with whey protein increased the adipose tissue leptin expression which is decreased in this obesity-prone mouse strain. These changes are likely to contribute to the inhibition of weight gain. The potential sensitisation of insulin signalling in adipose tissue together with the less lipotoxic and diabetogenic hepatic lipid profile suggest a novel mechanistic link to explain why increased dairy intake is associated with a lower prevalence of metabolic syndrome and type 2 diabetes in epidemiological studies. Taken together, the intake of a high-calcium diet with dairy proteins has a body weight lowering effect in high-fat-fed C57Bl/6J mice. High-calcium diets containing whey protein prevent weight gain and enhance weight loss, α-lactalbumin being the most effective whey protein fraction. Whey proteins and calcium have also beneficial effects on hepatic lipid profile and adipose tissue gene expression, which suggest a novel mechanistic link to explain the epidemiological findings on dairy intake and metabolic syndrome. The clinical relevance of these findings and the precise mechanisms of action remain an intriguing field of future research.

ADAMs, Osteoclastogenesis and Bone Destruction in the Loosening of the Totally Replaced Hip

Total hip replacement is the golden standard treatment for severe osteoarthritis refractory for conservative treatment. Aseptic loosening and osteolysis are the major long-term complications after total hip replacement. Foreign body giant cells and osteoclasts are locally formed around aseptically loosening implants from precursor cells by cell fusion. When the foreign body response is fully developed, it mediates inflammatory and destructive host responses, such as collagen degradation. In the present study, it was hypothesized that the wear debris and foreign body inflammation are the forces driving local osteoclast formation, peri-implant bone resorption and enhanced tissue remodeling. Therefore the object was to characterize the eventual expression and the role of fusion molecules, ADAMs (an abbreviation for A Disintegrin And Metalloproteinase, ADAM9 and ADAM12) in the fusion of progenitor cells into multinuclear giant cells. For generation of such cells, activated macrophages trying to respond to foreign debris play an important role. Matured osteoclasts together with activated macrophages mediate bone destruction by secreting protons and proteinases, including matrix metalloproteinases (MMPs) and cathepsin K. Thus this study also assessed collagen degradation and its relationship to some of the key collagenolytic proteinases in the aggressive synovial membrane-like interface tissue around aseptically loosened hip replacement implants. ADAMs were found in the interface tissues of revision total hip replacement patients. Increased expression of ADAMs at both transcriptional and translational levels was found in synovial membrane-like interface tissue of revision total hip replacement (THR) samples compared with that in primary THR samples. These studies also demonstrate that multinucleate cell formation from monocytes by stimulation with macrophage-colony stimiulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL) is characterized by time dependent changes of the proportion of ADAMs positive cells. This was observed both in the interface membrane in patients and in two different in vitro models. In addition to an already established MCS-F and RANKL driven model, a new virally (parainfluenza 2) driven model (of human salivary adenocarcinoma (HSY) cells or green monkey kidney (GMK) cells) was developed to study various fusion molecules and their role in cell fusion in general. In interface membranes, collagen was highly degraded and collagen degradation significantly correlated with the number of local cells containing collagenolytic enzymes, particularly cathepsin K. As a conclusion, fusion molecules ADAM9 and ADAM12 seem to be dynamically involved in cell-cell fusion processes and multinucleate cell formation. The highly significant correlation between collagen degradation and collagenolytic enzymes, particularly cathepsin K, indicates that the local acidity of the interface membrane in the pathologic bone and soft tissue destruction. This study provides profound knowledge about cell fusion and mechanism responsible for aseptic loosening as well as increases knowledge helpful for prevention and treatment.

The Mechanisms of Hedgehog Signal Transduction

Studies in both vertebrates and invertebrates have identified proteins of the Hedgehog (Hh) family of secreted signaling molecules as key organizers of tissue patterning. Initially discovered in Drosophila in 1992, Hh family members have been discovered in animals with body plans as diverse as those of mammals, insects and echinoderms. In humans three related Hh genes have been identified: Sonic, Indian and Desert hedgehog (Shh, Ihh and Dhh). Transduction of the Hh signal to the cytoplasm utilizes an unusual mechanism involving consecutive repressive interactions between Hh and its receptor components, Patched (Ptc) and Smoothened (Smo). Several cytoplasmic proteins involved in Hh signal transduction are known in Drosophila, but mammalian homologs are known only for the Cubitus interruptus (Ci) transcription factor (GLI(1-3)) and for the Ci/GLI-associated protein, Suppressor of Fused (Su(fu)). In this study I analyzed the mechanisms of how the Hh receptor Ptc regulates the signal transducer Smo, and how Smo relays the Shh signal from the cell surface to the cytoplasm ultimately leading to the activation of GLI transcription factors. In Drosophila, the kinesin-like protein Costal2 (Cos2) is required for suppression of Hh target gene expression in the absence of ligand, and loss of Cos2 causes embryonic lethality. Cos2 acts by bridging Smo to the Ci. Another protein, Su(Fu) exerts a weak suppressive influence on Ci activity and loss of Su(Fu) causes subtle changes in Drosophila wing pattern. This study revealed that domains in Smo that are critical for Cos2 binding in Drosophila are dispensable for mammalian Smo function. Furthermore, by analyzing the function of Su(Fu) and the closest mouse homologs of Cos2 by protein overexpression and RNA interference I found that inhibition of the Hh response pathway in the absence of ligand does not require Cos2 activity, but instead critically depends on the activity of Su(Fu). These results indicate that a major change in the mechanism of action of a conserved signaling pathway occurred during evolution, probably through phenotypic drift made possible by the existence in some species of two parallel pathways acting between the Hh receptor and the Ci/GLI transcription factors. In a second approach to unravel Hh signaling we cloned > 90% of all human full-length protein kinase cDNAs and constructed the corresponding kinase-activity deficient mutants. Using this kinome resource as a screening tool, two kinases, MAP3K10 and DYRK2 were found to regulate Shh signaling. DYRK2 directly phosphorylated and induced the proteasome dependent degradation of the key Hh-pathway regulated transcription factor, GLI2. MAP3K10, in turn, affected GLI2 indirectly by modulating the activity of DYRK2.