Differential and defective transcription of koala retrovirus indicates the complexity of host and virus evolution.

Koala retrovirus (KoRV) is unique amongst endogenous (inherited) retroviruses in that its incorporation to the host genome is still active, providing an opportunity to study what drives this fundamental process in vertebrate genome evolution. Animals in the southern part of the natural range of koalas were previously thought to be either virus-free or to have only exogenous variants of KoRV with low rates of KoRV-induced disease. In contrast, animals in the northern part of their range universally have both endogenous and exogenous KoRV with very high rates of KoRV-induced disease such as lymphoma. In this study we use a combination of sequencing technologies, Illumina RNA sequencing of 'southern' (south Australian) and 'northern' (SE QLD) koalas and CRISPR enrichment and nanopore sequencing of DNA of 'southern' (South Australian and Victorian animals) to retrieve full-length loci and intregration sites of KoRV variants. We demonstrate that koalas that tested negative to the KoRV pol gene qPCR, used to detect replication-competent KoRV, are not in fact KoRV-free but harbour defective, presumably endogenous, 'RecKoRV' variants that are not fixed between animals. This indicates that these populations have historically been exposed to KoRV and raises questions as to whether these variants have arisen by chance or whether they provide a protective effect from the infectious forms of KoRV. This latter explanation would offer the intriguing prospect of being able to monitor and selectively breed for disease resistance to protect the wild koala population from KoRV-induced disease.

A new look at the origins of gibbon ape leukemia virus.

Is the origin of gibbon ape leukemia virus (GALV) human after all? When GALV was discovered and found to cause neoplastic disease in gibbons, it stimulated a great deal of research including investigations into the origins of this virus. A number of publications have suggested that the GALV progenitor was a retrovirus present in one of several species of South East Asian rodents that had close contact with captive gibbons. However, there are no published retroviral sequences from any South East Asian species to support this view. Here we present an alternative hypothesis that the origin of GALV is a virus closely related to Melomys burtoni retrovirus, and that this virus infected human patients in Papua New Guinea from whom biological material was obtained or in some way contaminated these samples. This material we propose contained infectious MbRV-related virus that was then unwittingly introduced into gibbons which subsequently developed GALV infections.

Identifying information needs among children and teens living with haemophilia.

Transitioning from one life stage to the next can be difficult, but for those living with a chronic condition, it can be even more challenging. Children and adolescents with haemophilia need help to manage transitions while dealing with the complications of their disorder. The National Haemophilia Foundation (NHF), headquartered in New York City, has an extensive information centre on bleeding disorders, but it was not clear how much material existed on the topic of transition. The objectives of this project were to (i) assess the availability of literature about transition for children and adolescents living with haemophilia, (ii) determine which transition issues were the most relevant and (iii) develop and test information products that would address those transition issues. An inventory of NHF's resources and an environmental scan over the Internet was performed. Focus groups were conducted to determine messaging. Video prototypes containing messages were created, tested by focus groups and revised. The literature search yielded limited information available on transition for children and adolescents with haemophilia. Results of the formative research indicated that adolescents wanted more information on sports participation and disclosure of their condition (e.g. to peers, teachers, coaches, health care providers). Video was found to be the preferred delivery format. Children and adolescents living with haemophilia need information to help them transition through life. As a result of this study, two educational products were produced, but several more are recommended to guide these individuals in making healthy transitions into adulthood.

Social competence in autism: A structural equation modeling approach.

Autistic individuals present with difficulties in social competence (e.g., navigating social interactions and fostering relationships). Clinical interventions widely target social cognition and social behavior, but there is inconsistent understanding of the underlying components of social competence. The present study used structural equation modeling to examine social cognition and social behavior and explore the relationship between these latent constructs. Autistic youth (ages 10-17; n = 219) and their caregivers participated in this study. Constructs of social cognition and social behavior were captured using caregiver-report and self-report rating scales, as well as observational measures and direct clinical assessments (e.g., NEPSY-II). Measurement models of social cognition and social behavior demonstrated adequate to good fit. Correlational models demonstrated adequate to poor fit, indicating latent constructs of social cognition and social behavior are not closely related in autistic youth. Exploratory examination of a subsample of male youth (n = 157) evidenced improved model fit of social behavior, specifically. Findings tease apart social cognition and social behavior as cohesive and separable constructs; results do not support a structural relationship between social cognition and social behavior. Noted treatment implications include consideration of how targeting social cognition and social behavior together or separately may support autistic youth's progress toward reaching their identified therapeutic goals and supporting their self-directed social development.

Vascular endothelium-specific overexpression of human catalase in cloned pigs.

The objective of this study was to develop transgenic Yucatan minipigs that overexpress human catalase (hCat) in an endothelial-specific manner. Catalase metabolizes hydrogen peroxide (H(2)O(2)), an important regulator of vascular tone that contributes to diseases such as atherosclerosis and preeclampsia. A large animal model to study reduced endothelium-derived H(2)O(2) would therefore generate valuable translational data on vascular regulation in health and disease. Yucatan minipig fetal fibroblasts stably co-transfected with human catalase (Tie2-hCat) and eGFP expression constructs were isolated into single-cell populations. The presence of the Tie2-hCat transgene in individual colonies of fibroblasts was determined by PCR. Transgenic fibroblasts were used for nuclear transfer into enucleated oocytes by electrofusion. A minimum of 140 cloned embryos were transferred per surrogate sow (n = 4). All four surrogates maintained pregnancies and piglets were delivered by cesarean section. Nine male piglets from three of the four litters carried the Tie2-hCat transgene. Expression of human catalase mRNA and overall elevated catalase protein in isolated umbilical endothelial cells from transgenic piglets were verified by RT-PCR and western blot, respectively, and endothelial localization was confirmed by immunohistochemistry. Increased enzymatic activity of catalase in transgenic versus wild-type endothelial cells was inferred based on significantly reduced levels of H(2)O(2) in culture. The similarities in swine and human cardiovascular anatomy and physiology will make this pig model a valuable source of information on the putative role of endothelium-derived H(2)O(2) in vasodilation and in the mechanisms underlying vascular health and disease.

Novel trypanosome Trypanosoma gilletti sp. (Euglenozoa: Trypanosomatidae) and the extension of the host range of Trypanosoma copemani to include the koala ( Phascolarctos cinereus).

Trypanosoma irwini was previously described from koalas and we now report the finding of a second novel species, T. gilletti, as well as the extension of the host range of Trypanosoma copemani to include koalas. Phylogenetic analysis at the 18S rDNA and gGAPDH loci demonstrated that T. gilletti was genetically distinct with a genetic distance (± s.e.) at the 18S rDNA locus of 2.7 ± 0.5% from T. copemani (wombat). At the gGAPDH locus, the genetic distance (± s.e.) of T. gilletti was 8.7 ± 1.1% from T. copemani (wombat). Trypanosoma gilletti was detected using a nested trypanosome 18S rDNA PCR in 3/139 (∼2%) blood samples and in 2/29 (∼7%) spleen tissue samples from koalas whilst T. irwini was detected in 72/139 (∼52%) blood samples and T. copemani in 4/139 (∼3%) blood samples from koalas. In addition, naturally occurring mixed infections were noted in 2/139 (∼1.5%) of the koalas tested.

Nuclear F-actin Cytology in Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma.

Oral cavity cancer has a low 5-y survival rate, but outcomes improve when the disease is detected early. Cytology is a less invasive method to assess oral potentially malignant disorders relative to the gold-standard scalpel biopsy and histopathology. In this report, we aimed to determine the utility of cytological signatures, including nuclear F-actin cell phenotypes, for classifying the entire spectrum of oral epithelial dysplasia and oral squamous cell carcinoma. We enrolled subjects with oral potentially malignant disorders, subjects with previously diagnosed malignant lesions, and healthy volunteers without lesions and obtained brush cytology specimens and matched scalpel biopsies from 486 subjects. Histopathological assessment of the scalpel biopsy specimens classified lesions into 6 categories. Brush cytology specimens were analyzed by machine learning classifiers trained to identify relevant cytological features. Multimodal diagnostic models were developed using cytology results, lesion characteristics, and risk factors. Squamous cells with nuclear F-actin staining were associated with early disease (i.e., lower proportions in benign lesions than in more severe lesions), whereas small round parabasal-like cells and leukocytes were associated with late disease (i.e., higher proportions in severe dysplasia and carcinoma than in less severe lesions). Lesions with the impression of oral lichen planus were unlikely to be either dysplastic or malignant. Cytological features substantially improved upon lesion appearance and risk factors in predicting squamous cell carcinoma. Diagnostic models accurately discriminated early and late disease with AUCs (95% CI) of 0.82 (0.77 to 0.87) and 0.93 (0.88 to 0.97), respectively. The cytological features identified here have the potential to improve screening and surveillance of the entire spectrum of oral potentially malignant disorders in multiple care settings.

Aminooxypentane-RANTES induces CCR5 internalization but inhibits recycling: a novel inhibitory mechanism of HIV infectivity.

CCR5, a chemokine receptor expressed on T cells and macrophages, is the principal coreceptor for M-tropic HIV-1 strains. Recently, we described an NH2-terminal modification of the CCR5 ligand regulated on activation, normal T cell expressed and secreted (RANTES), aminooxypentane-RANTES (AOP-RANTES), that showed potent inhibition of macrophage infection by HIV-1 under conditions where RANTES was barely effective. To investigate the mechanism of AOP-RANTES inhibition of HIV infectivity we examined the surface expression of CCR5 using a monoclonal anti-CCR5 antibody, MC-1. We demonstrate that AOP-RANTES rapidly caused >90% decrease in cell surface expression of CCR5 on lymphocytes, monocytes/ macrophages, and CCR5 transfected Chinese hamster ovary (CHO) cells. RANTES also caused a loss of cell surface CCR5, although its effect was less than with AOP-RANTES. Significantly, AOP-RANTES inhibited recycling of internalized CCR5 to the cell surface, whereas RANTES did not. When peripheral blood mononuclear cells are cultured for prolonged periods of time in the presence of RANTES, CCR5 expression is comparable to that seen on cells treated with control medium, whereas there is no CCR5 surface expression on cells cultured in the presence of AOP-RANTES. Immunofluorescence indicated that both AOP-RANTES and RANTES induced downmodulation of cell surface CCR5, and that the receptor was redistributed into endocytic organelles containing the transferrin receptor. When RANTES was removed, the internalized receptor was recycled to the cell surface; however, the receptor internalized in the presence of AOP-RANTES was retained in endosomes. Using human osteosarcoma (GHOST) 34/CCR5 cells, the potency of AOP-RANTES and RANTES to inhibit infection by the M-tropic HIV-1 strain, SF 162, correlated with the degree of downregulation of CCR5 induced by the two chemokines. These differences between AOP-RANTES and RANTES in their effect on receptor downregulation and recycling suggest a mechanism for the potent inhibition of HIV infection by AOP-RANTES. Moreover, these results support the notion that receptor internalization and inhibition of receptor recycling present new targets for therapeutic agents to prevent HIV infection.

piggyBac-like elements in the pink bollworm, Pectinophora gossypiella.

A transgenic line of the pink bollworm, Pectinophora gossypiella, a key lepidopteran cotton pest, was generated previously using the piggyBac transposon IFP2 from Trichoplusia ni. Here we identified an endogenous piggyBac-like element (PLE), designated as PgPLE1, in the pink bollworm. A putatively intact copy of PgPLE1 (PgPLE1.1) presents the canonical features of PLE: inverted terminal repeats with three C/G residues at the extreme ends, inverted subterminal repeats, TTAA target site and an open reading frame encoding transposase with 68% similarity to IFP2. Vectorette PCR revealed large variation in the insertion sites of PgPLE1 amongst worldwide populations, indicating the potential mobility of PgPLE1. The PgPLE1 was undetectable in the genome of Pectinophora endema, implying the recent invasion of PgPLE1 after the divergence of these two closely related species.

Pathological Findings in Koala Retrovirus-positive Koalas (Phascolarctos cinereus) from Northern and Southern Australia.

Koala retrovirus (KoRV) infection shows differences in prevalence and load between northern and southern Australian koala populations; however, the effect of this on diseases such as lymphoma and chlamydial disease is unclear. This study compared clinicopathological findings, haematology and splenic lymphoid area of KoRV-positive koalas from northern (Queensland [Qld], n = 67) and southern (South Australia [SA], n = 92) populations in order to provide further insight into KoRV pathogenesis. Blood was collected for routine haematology and for measurement of KoRV proviral load by quantitative polymerase chain reaction (qPCR). Plasma samples were assessed for KoRV viral load by reverse transcriptase qPCR and conjunctival and cloacal swabs were collected for measurement of the load of Chlamydia pecorum (qPCR). During necropsy examination, spleen was collected for lymphoid area analysis. Lymphoma was morphologically similar between the populations and occurred in koalas with the highest KoRV proviral and viral loads. Severe ocular chlamydial disease was observed in both populations, but urinary tract disease was more severe in Qld, despite similar C. pecorum loads. No associations between KoRV and chlamydial disease severity or load were observed, except in SA where viral load correlated positively with chlamydial disease severity. In both populations, proviral and viral loads correlated positively with lymphocyte and metarubricyte counts and correlated negatively with erythrocyte and neutrophil counts. Splenic lymphoid area was correlated positively with viral load. This study has shown further evidence for KoRV-induced oncogenesis and highlighted that lymphocytes and splenic lymphoid tissue may be key sites for KoRV replication. However, KoRV infection appears to be highly complex and continued investigation is required to fully understand its pathogenesis.

Granites: Relation of Properties in situ to Laboratory Measurements.

The velocity of compressional waves and electrical resistivity in granite in situ measured in two 3-kilometer boreholes exhibits very little variation with depth, in contrast with the variation predicted from laboratory measurements on dry samples. These observations can be explained either by the absence of small open cracks in the rocks in situ or by the effects of complete saturation with water. The seismic velocities of many granites at shallow depths in the earth's crust may be significantly larger than was previously believed. Other properties are also affected; correction for the effect of cracks on thermal conductivity raises the average heat flow in shield areas by as much as 20 percent.

The 25-km Discontinuity: Implications for Lunar History.

The lunar velocity profile and laboratory data on terrestrial and lunar rocks are constraints on models of lunar history. They show that shock-induced microcracks are absent from the rocks present in the moon today at depths of 25 to 60 kilometers. All possible causes of this observation are examined, and the most likely explanations are that either the rocks at depths of 25 to 60 kilometers formed after the major impacts ceased or the microcracks have annealed at temperatures of about 600 degrees C over geologically long times.

Elastic wave velocities of lunar samples at high pressures and their geophysical implications.

Ultrasonic measurement of P and S velocities of Apollo 11 lunar samples 10020, 10057, and 10065 to 5 kilobars pressure at room temperature shows a pronounced increase of velocity (as much as twofold) for the first 2 kilobars. The travel times predicted from the velocity-depth curve of sample 10057 are consistent with the results of the Apollo 12 seismic experiments. At pressures below 200 bars, the samples are highly attenuating; for both P and S waves, the value of Q is about 10.

Thermal diffusivity and conductivity of lunar material.

The thermal diffusivity and conductivity of type C lunar samples returned by Apollo 11 are lower and less dependent on temperature than those of type A samples. The thermal properties of both types are lower than the corresponding properties of normal terrestrial rocks.

Potent inhibition of HIV-1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist.

The chemokine receptors CXCR4 and CCR5 have recently been shown to act as coreceptors, in concert with CD4, for human immunodeficiency virus-type 1 (HIV-1) infection. RANTES and other chemokines that interact with CCR5 and block infection of peripheral blood mononuclear cell cultures inhibit infection of primary macrophages inefficiently at best. If used to treat HIV-1-infected individuals, these chemokines could fail to influence HIV replication in nonlymphocyte compartments while promoting unwanted inflammatory side effects. A derivative of RANTES that was created by chemical modification of the amino terminus, aminooxypentane (AOP)-RANTES, did not induce chemotaxis and was a subnanomolar antagonist of CCR5 function in monocytes. It potently inhibited infection of diverse cell types (including macrophages and lymphocytes) by nonsyncytium-inducing, macrophage-tropic HIV-1 strains. Thus, activation of cells by chemokines is not a prerequisite for the inhibition of viral uptake and replication. Chemokine receptor antagonists like AOP-RANTES that achieve full receptor occupancy at nanomolar concentrations are strong candidates for the therapy of HIV-1-infected individuals.

A broad-spectrum chemokine antagonist encoded by Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus encodes a chemokine called vMIP-II. This protein displayed a broader spectrum of receptor activities than any mammalian chemokine as it bound with high affinity to a number of both CC and CXC chemokine receptors. Binding of vMIP-II, however, was not associated with the normal, rapid mobilization of calcium from intracellular stores; instead, it blocked calcium mobilization induced by endogenous chemokines. In freshly isolated human monocytes the virally encoded vMIP-II acted as a potent and efficient antagonist of chemotaxis induced by chemokines. Because vMIP-II could inhibit cell entry of human immunodeficiency virus (HIV) mediated through CCR3 and CCR5 as well as CXCR4, this protein may serve as a lead for development of broad-spectrum anti-HIV agents.

Abnormal digital neurovascular response to local heating in systemic sclerosis.

OBJECTIVES: To investigate neurovascular dysfunction using the axon reflex-dependent hyperaemia (initial peak of skin local heating response) in fingers of patients with SSc or primary RP. METHODS: Ten healthy subjects were initially enrolled to compare axon reflex-dependent thermal hyperaemia between the finger and forearm cutaneous circulations. Then, 10 patients with primary RP and 16 patients with SSc participated in a similar protocol focusing on the finger circulation only. Lidocaine/prilocaine cream was applied for 1 h to produce local blockade of cutaneous sensory nerves. After lidocaine/prilocaine pre-treatment, laser Doppler probes were heated from skin temperature to 42 degrees C for 30 min, and 44 degrees C for 5 min to achieve maximal skin blood flow. Data were expressed as a percentage of maximal cutaneous vascular conductance. RESULTS: In healthy volunteers, we observed a significantly higher initial peak on the finger compared with the forearm, with both responses blunted following topical anaesthesia. In primary RP patients, we observed a decreased initial peak following lidocaine/prilocaine pre-treatment in the finger circulation [96.7% (33.4) vs 75.9% (29.5) with anaesthesia, P = 0.02]. In contrast, pre-treatment did not alter the initial peak in patients with SSc. A minute-by-minute analysis showed no delay of the initial peak. CONCLUSIONS: We show an abnormal digital neurovascular response to local heating in SSc. Thermal hyperaemia could be monitored as a clinical test for neurovascular function in SSc. Further studies are required to test whether the abnormal digital neurovascular response correlates to the degree of peripheral vascular involvement.

Extended-spectrum beta-lactamase (ESBL)-producing enterobacteria: factors associated with infection in the community setting, Auckland, New Zealand.

We aimed to document the epidemiology of extended-spectrum beta-lactamase (ESBL)-producing enterobacteria in the Auckland community and identify factors associated with infection using a case-control study design. ESBL-producing enterobacteria were isolated from 107 infected patients, for which demographic and clinical data were available for 98 cases (92%). Escherichia coli was the predominant organism (82%), with urine as the commonest source (97%). Compared with a control group infected with ESBL-negative enterobacteria, factors significantly associated with infection on univariate analysis were: living in a residential care home (RCH); recent admission to hospital 'M'; recent antibiotic use; older age (>75 years); presence of a urinary catheter; and a history of comorbid chronic obstructive pulmonary disease (COPD), cardiovascular disease, neurological disease or recurrent urinary tract infection. On multivariate analysis, residence in RCH and COPD remained significant associations. Pulsed-field gel electrophoresis typing of the ESBL-producing E. coli identified a common strain. We concluded that residence in RCH and a history of COPD are significant associations with ESBL-producing enterobacterial infection in the Auckland community. Several spatial clusters in RCHs and a common strain suggest point-source outbreaks. A substantial number of community cases did not live in an RCH nor had been recently hospitalised, suggesting the independent generation of ESBL-producing enterobacteria in the broader community.

Identification of stable reference genes for quantitative PCR in koalas.

To better understand host and immune response to diseases, gene expression studies require identification of reference genes with stable expression for accurate normalisation. This study describes the identification and testing of reference genes with stable expression profiles in koala lymph node tissues across two genetically distinct koala populations. From the 25 most stable genes identified in transcriptome analysis, 11 genes were selected for verification using reverse transcription quantitative PCR, in addition to the commonly used ACTB and GAPDH genes. The expression data were analysed using stable genes statistical software - geNorm, BestKeeper, NormFinder, the comparative ΔCt method and RefFinder. All 13 genes showed relative stability in expression in koala lymph node tissues, however Tmem97 and Hmg20a were identified as the most stable genes across the two koala populations.

Lymphoma, Koala Retrovirus Infection and Reproductive Chlamydiosis in a Koala (Phascolarctos cinereus).

Koala retrovirus (KoRV) infection, thought to be associated with lymphoid neoplasia, and Chlamydia pecorum-related ocular and urogenital disease are both highly prevalent in eastern Australian koala (Phascolarctos cinereus) populations. However, in South Australian koalas, little is known about KoRV infection and C. pecorum-associated disease. We report the first South Australian case of lymphoma in a KoRV-A-positive female koala also affected by severe reproductive chlamydiosis. The koala was from the Mount Lofty Ranges population and was presented with hindlimb lameness. Clinical examination identified right stifle crepitus, enlarged superficial lymph nodes and paraovarian cysts. Necropsy examination revealed extensive cartilage degeneration and loss over the medial femoral condyle, solid femoral bone marrow, mesenteric and ovarian tumours, paraovarian cysts and purulent metritis. Histopathology confirmed lymphoma in the bone marrow, mesenteric lymph nodes and ovary, with infiltration and parenchymal effacement in the pancreas, adrenal glands and other tissues. Lymphoma, KoRV and chlamydiosis are being investigated further in this population.