Disruption of the non-canonical Wnt gene PRICKLE2 leads to autism-like behaviors with evidence for hippocampal synaptic dysfunction.

Autism spectrum disorders (ASDs) have been suggested to arise from abnormalities in the canonical and non-canonical Wnt signaling pathways. However, a direct connection between a human variant in a Wnt pathway gene and ASD-relevant brain pathology has not been established. Prickle2 (Pk2) is a post-synaptic non-canonical Wnt signaling protein shown to interact with post-synaptic density 95 (PSD-95). Here, we show that mice with disruption in Prickle2 display behavioral abnormalities including altered social interaction, learning abnormalities and behavioral inflexibility. Prickle2 disruption in mouse hippocampal neurons led to reductions in dendrite branching, synapse number and PSD size. Consistent with these findings, Prickle2 null neurons show decreased frequency and size of spontaneous miniature synaptic currents. These behavioral and physiological abnormalities in Prickle2 disrupted mice are consistent with ASD-like phenotypes present in other mouse models of ASDs. In 384 individuals with autism, we identified two with distinct, heterozygous, rare, non-synonymous PRICKLE2 variants (p.E8Q and p.V153I) that were shared by their affected siblings and inherited paternally. Unlike wild-type PRICKLE2, the PRICKLE2 variants found in ASD patients exhibit deficits in morphological and electrophysiological assays. These data suggest that these PRICKLE2 variants cause a critical loss of PRICKLE2 function. The data presented here provide new insight into the biological roles of Prickle2, its behavioral importance, and suggest disruptions in non-canonical Wnt genes such as PRICKLE2 may contribute to synaptic abnormalities underlying ASDs.

Treatment for older men with fractures.

UNLABELLED: Less than 10% of men receive osteoporosis treatment, even after a fracture. A study of 17,683 men revealed that older men, those with spinal fractures, and those taking steroids or antidepressants are more likely to receive treatment after a fracture. Seeing a primary care physician also increases osteoporosis treatment rates. INTRODUCTION: In 2000, the FDA approved bisphosphonates for the treatment of osteoporosis in men. The purpose of this study is to estimate the frequency of bisphosphonate therapy within 12 months following a fracture and describe patient/physician factors associated with treatment. METHODS: Health insurance claims for 17,683 men ≥ 65 years of age, who had a claim for an incident fracture from 2000 to 2005, were followed for at least 6 months post-fracture for the initiation of treatment with a bisphosphonate. Patient characteristics, diagnostic procedures, therapies, co-morbidities, and provider characteristics were compared for men who received treatment with those who did not. RESULTS: Eight percent of men (n = 1,434) received bisphosphonate therapy. Overall treatment increased from 7% in 2001 to 9% in 2005 (p < 0.001). Treatment for hip fractures remained at 7% (p = 0.747). Treatment increased with age: 6% in men aged 65-69 compared to 11.6% in men aged 85-89 (p < 0.001). Factors associated with treatment included: diagnosis of osteoporosis (OR = 8.8; 95% CI, 7.7, 10.4), glucocorticoid therapy (OR = 3.2; 95% CI, 2.4, 4.3), bone mineral density measurement (OR = 3.4; 95% CI, 2.9, 4.0), and antidepressant therapy with tricyclics (OR = 2.0; 95% CI, 1.2, 3.5) or selective serotonin reuptake inhibitors (OR = 1.7; 95% CI, 1.3, 2.4). Men with vertebral fractures (OR = 2.2; 95% CI, 1.8, 2.6) and men seen by primary physicians (OR = 2.6; 95% CI, 2.3, 3.1) were more likely to receive treatment. CONCLUSIONS: Less than 10% of men received bisphosphonate therapy following a low-impact fracture. Men with a primary physician were more likely to receive bisphosphonate therapy; however, <25% of men were seen by a primary physician.

Tracking the Migraine Cycle Using Visual Tasks.

There are a number of reports that perceptual, electrophysiological and imaging measures can track migraine periodicity. As the electrophysiological and imaging research requires specialist equipment, it has few practical applications. This study sought to track changes in performance on four visual tasks over the migraine cycle. Coherence thresholds were measured for two motion and two orientation tasks. The first part of the study confirmed that the data obtained from an online study produced comparable results to those obtained under controlled laboratory conditions. Thirteen migraine with aura, 12 without aura, and 12 healthy controls participated. The second part of the study showed that thresholds for discriminating vertical coherent motion varied with the migraine cycle for a majority of the participants who tested themselves multiple times (four with aura, seven without). Performance improved two days prior to a migraine attack and remained improved for two days afterwards. This outcome is as expected from an extrapolation of earlier electrophysiological research. This research points to the possibility of developing sensitive visual tests that patients can use at home to predict an impending migraine attack and so take steps to try to abort it or, if it is inevitable, to plan their lives around it.

Changes in induced hues at low luminance and following dark adaptation suggest rod-cone interactions may differ for luminance increments and decrements.

Color contrast describes the influence of one color on the perception of colors in neighboring areas. This study addressed two issues: (1) the accurate representation of the color changes; (ii) the underlying visual mechanisms. Observers matched the hue that was induced in a neutral square when it was set in one of four standard colored surrounds: "red" (+L(-M) relative to neutral), "green" (-L(+M)), "purple" (+S), and "yellow" (-S). The standard and matching displays were viewed haploscopically. The standard neutral square was either a luminance increment, or decrement, both of which appeared the complementary color to the surrounds in which they were inset. In Experiment 1, the surround luminance in each eye's display was either equal, at 18 cd x m(-2), or the match surround luminance was reduced to 2.5 cd x m(-2). The matches with equal surround luminances could be represented as vector shifts in a logarithmic MacLeod-Boynton (r, b) chromaticity diagram, as described previously (Shepherd, 1997, 1999). The low luminance matches were, however, displaced further from neutral, as if larger chromatic differences were needed. The precise direction of the displacements differed for luminance increments and decrements: the red, green and yellow decrement matches were also displaced vertically downwards in the MacLeod-Boynton diagram. In Experiment 2, dark-adapting before setting repeat color matches displaced the decrement matches vertically, but did not affect the increment matches. Thus, rod intrusion in S-cone pathways may have boosted the S-cone signal for the lowest luminance decrement matches in Experiment 1 and account for the vertical shift in MacLeod-Boynton co-ordinates. The distinct pattern of displacements for low luminance increments and decrements may be explained if the match is set at a cone-opponent, rather than a cone contrast, site and if rod signals have an input only to S-cone decrement, perhaps S-OFF, pathways.

Parathyroid hormone-related peptide activates and modulates TRPV1 channel in human DRG neurons.

Parathyroid hormone-related peptide (PTHrP) is associated with advanced tumor growth and metastasis, especially in breast, prostate and myeloma cancers that metastasize to bones, resulting in debilitating chronic pain conditions. Our recent studies revealed that the receptor for PTHrP, PTH1R, is expressed in mouse DRG sensory neurons, and its activation leads to flow-activation and modulation of TRPV1 channel function, resulting in peripheral heat and mechanical hypersensitivity. In order to verify the translatability of our findings in rodents to humans, we explored whether this signalling axis operates in primary human DRG sensory neurons. Analysis of gene expression data from recently reported RNA deep sequencing experiments performed on mouse and human DRGs reveals that PTH1R is expressed in DRG and tibial nerve. Furthermore, exposure of cultured human DRG neurons to PTHrP leads to slow-sustained activation of TRPV1 and modulation of capsaicin-induced channel activation. Both activation and modulation of TRPV1 by PTHrP were dependent on PKC activity. Our findings suggest that functional PTHrP/PTH1R-TRPV1 signalling exists in human DRG neurons, which could contribute to local nociceptor excitation in the vicinity of metastatic bone tumor microenvironment.

Local and global motion after-effects are both enhanced in migraine, and the underlying mechanisms differ across cortical areas.

Visual after-effects are illusions that occur after prolonged viewing of visual displays (pattern adaptation). The motion after-effect (MAE), for example, is an illusory impression of motion that is seen after viewing moving displays. After-effects have been used extensively in basic vision research as well as in clinical settings, and have been reported to be enhanced in migraine. Pattern adaptation is a cortical phenomenon that reflects both cellular mechanisms acting within individual neurons and specific interactions between groups of neurons activated by the adapting display. A remarkable feature of the MAE is that its duration is only slightly reduced if a delay is inserted between the end of the adaptation and the test display ('storage'). The reduction is consistent with recovery of the cellular component, and the residual with network changes that are maintained during the delay. This study aimed (i) to assess explanations for prolonged MAEs in migraine by teasing apart the proposed cellular and network components of adaptation using storage; (ii) to determine the extent of cortical abnormality in migraine using local and global MAEs, which reflect adaptation at different stages of the visual system. Fifty migraine (22 with, 28 without aura) and 50 control participants adapted to motion before viewing a stationary or dynamic (random motion) test, which consequently appeared to move in the opposite direction (local and global MAEs, respectively). Half of the trials included a delay between the adapting and test displays. The results extend those reported previously, as both local and global MAEs lasted longer in migraine compared with the control group. Global MAEs survived delays almost completely for both groups, whereas local MAEs were reduced to a greater extent in migraine. There were no significant differences between migraine subgroups classified according to the presence or absence of visual aura. These results suggest that cellular recovery is slowed in migraine for early but not later visual cortical areas. Sustained network changes following adaptation are implicated across cortical areas. Differences between people with and without migraine on various measures of visual perception have been attributed to abnormal cortical processing in migraine, variously described by hyperexcitability, heightened responsiveness and/or a lack of intra-cortical inhibition. The results are not consistent with hyperexcitability resulting from a lack of inhibition in migraine, but are consistent with extended suppression of intra-cortical excitation. The implications of these results for alternative models of hyperexcitability are discussed.

VIDA: a virus database system for the organization of animal virus genome open reading frames.

VIDA is a new virus database that organizes open reading frames (ORFs) from partial and complete genomic sequences from animal viruses. Currently VIDA includes all sequences from GenBank for Herpesviridae, Coronaviridae and Arteriviridae. The ORFs are organized into homologous protein families, which are identified on the basis of sequence similarity relationships. Conserved sequence regions of potential functional importance are identified and can be retrieved as sequence alignments. We use a controlled taxonomical and functional classification for all the proteins and protein families in the database. When available, protein structures that are related to the families have also been included. The database is available for online search and sequence information retrieval at http://www.biochem.ucl.ac.uk/bsm/virus_database/ VIDA.html.

A rapid classification protocol for the CATH Domain Database to support structural genomics.

In order to support the structural genomic initiatives, both by rapidly classifying newly determined structures and by suggesting suitable targets for structure determination, we have recently developed several new protocols for classifying structures in the CATH domain database (http://www.biochem.ucl.ac.uk/bsm/cath). These aim to increase the speed of classification of new structures using fast algorithms for structure comparison (GRATH) and to improve the sensitivity in recognising distant structural relatives by incorporating sequence information from relatives in the genomes (DomainFinder). In order to ensure the integrity of the database given the expected increase in data, the CATH Protein Family Database (CATH-PFDB), which currently includes 25,320 structural domains and a further 160,000 sequence relatives has now been installed in a relational ORACLE database. This was essential for developing more rigorous validation procedures and for allowing efficient querying of the database, particularly for genome analysis. The associated Dictionary of Homologous Superfamilies [Bray,J.E., Todd,A.E., Pearl,F.M.G., Thornton,J.M. and Orengo,C.A. (2000) Protein Eng., 13, 153-165], which provides multiple structural alignments and functional information to assist in assigning new relatives, has also been expanded recently and now includes information for 903 homologous superfamilies. In order to improve coverage of known structures, preliminary classification levels are now provided for new structures at interim stages in the classification protocol. Since a large proportion of new structures can be rapidly classified using profile-based sequence analysis [e.g. PSI-BLAST: Altschul,S.F., Madden,T.L., Schaffer,A.A., Zhang,J., Zhang,Z., Miller,W. and Lipman,D.J. (1997) Nucleic Acids Res., 25, 3389-3402], this provides preliminary classification for easily recognisable homologues, which in the latest release of CATH (version 1.7) represented nearly three-quarters of the non-identical structures.

Prediction of the location and type of beta-turns in proteins using neural networks.

A neural network has been used to predict both the location and the type of beta-turns in a set of 300 nonhomologous protein domains. A substantial improvement in prediction accuracy compared with previous methods has been achieved by incorporating secondary structure information in the input data. The total percentage of residues correctly classified as beta-turn or not-beta-turn is around 75% with predicted secondary structure information. More significantly, the method gives a Matthews correlation coefficient (MCC) of around 0.35, compared with a typical MCC of around 0.20 using other beta-turn prediction methods. Our method also distinguishes the two most numerous and well-defined types of beta-turn, types I and II, with a significant level of accuracy (MCCs 0.22 and 0.26, respectively).

Detection of unintegrated HIV type 1 DNA in cell culture and clinical peripheral blood mononuclear cell samples: correlation to disease stage.

This article reports on the development of PCR as a sensitive method of detecting both linear and circular forms of HIV-1 unintegrated viral DNA (UVD). The method was developed in a cell line study designed to follow the sequential synthesis of these forms over time. In all T lymphoid lineage cell lines, the full-length linear UVD (LUVD) was synthesized prior to both 1 and 2 LTR forms of circular UVD (CUVD), although all forms were detected by 12 hr postinoculation. Analysis of unstimulated PBMC samples from HIV-positive patients showed a significant difference in the presence of detectable CUVD forms and CDC groups II and IV (p < 0.001) and CDC groups III and IV (p < 0.001). No significance was demonstrated between CDC groups II and III (p > 0.5), linking the presence of CUVD forms to clinical disease and immunodeficiency. We propose that circular unintegrated forms of HIV-1 DNA may play a role in the development of acquired immunodeficiency syndrome.

Viremia and antibody response of small African and laboratory animals to Crimean-Congo hemorrhagic fever virus infection.

Eleven species of small African wild mammals, laboratory rabbits, guinea pigs, and Syrian hamsters were infected with Crimean-Congo hemorrhagic fever (CCHF) virus. Low-titered viremia followed by development of antibody was observed in scrub hares (Lepus saxatilis), Cape ground squirrels (Xerus inauris), red veld rats (Aethomys chrysophilus), white tailed rats (Mystromys albicaudatus), bushveld gerbils (Tatera leucogaster), striped mice (Rhabdomys pumilio), and guinea pigs. The maximum viremic titer in 4 scrub hares was 10(1.7-4.2) 50% mouse lethal doses/ml. Viremia was detected in 1/17 infected laboratory rabbits. Antibody response was only detected in South African hedgehogs (Atelerix frontalis), highveld gerbils (T. brantsii), Namaqua gerbils (Desmodillus auricularis), 2 species of multimammate mouse (Mastomys natalensis and M. coucha), and Syrian hamsters. The results of the study indicate that a proportion of infected scrub hares develop CCHF viremia of an intensity shown in the Soviet Union to be sufficient for infection of feeding immature ixodid ticks, but that South African hedgehogs and wild rodents are unlikely to be of importance as maintenance hosts of the virus in southern Africa.

Experimental studies on the replication and transmission of Crimean-Congo hemorrhagic fever virus in some African tick species.

Seven African tick species were studied as potential vectors of Crimean-Congo hemorrhagic fever (CCHF) virus. Engorged nymphae of 4 ixodid species, Hyalomma marginatum rufipes, H. truncatum, Rhipicephalus evertsi mimeticus, and Amblyomma hebraeum, were inoculated intracoelomically with CCHF virus and assayed for virus content at varying times post-inoculation. The virus replicated in all 4 species, reaching maximum titers of 4.6-5.5(10) fluorescence focus units per ml on days 5-9 post-inoculation. Virus titers declined up to the molt, but increased slightly on emergence of adult ticks. Thereafter, virus titers declined progressively, but infectivity could still be detected in adult ticks for up to 205 days post-inoculation. Groups of H. m. rufipes, H. truncatum, and R.e. mimeticus infected adults were fed on susceptible sheep and successfully transmitted CCHF infection. CCHF virus was not isolated from pools of the larval and nymphal progeny of the female ticks nor did the larvae transmit infection to guinea pigs by bite. CCHF virus failed to replicate in adults and nymphae of 3 argasid tick species, Argas walkerae, Ornithodorus porcinus porcinus, and O. savignyi, after intracoelomic inoculation and could be reisolated from the ticks no later than 1 day post-inoculation. The results suggest that all ixodid ticks are capable of transmitting CCHF virus but argasid ticks do not appear to be capable of serving as vectors.

Preparation and use of monoclonal antibodies for identifying Crimean-Congo hemorrhagic fever virus.

Seven monoclonal antibodies were prepared against a South African strain of Crimean-Congo hemorrhagic fever (CCHF) virus and were found to be directed against viral nucleocapsid protein. Five of the monoclonal antibodies reacted to high titer in indirect immunofluorescence (IF) tests and enzyme-linked immunosorbent assays (ELISA) with 22 strains of CCHF virus and failed to cross-react with the closest antigenic relative of CCHF, Hazara virus, or with 4 other nairoviruses which need to be distinguished from CCHF virus in Africa. These antibodies, used in the IF technique, readily detected antigens induced by all strains of CCHF virus included in the study in cell culture monolayers and mouse brain tissue, which represent the systems commonly used for isolation of CCHF virus. The IF technique with monoclonal antibodies constitutes a rapid and specific means of identifying newly isolated strains of CCHF virus.

Antibody to Crimean-Congo hemorrhagic fever virus in wild mammals from southern Africa.

Crimean-Congo hemorrhagic fever (CCHF) virus is becoming increasingly recognized as an important human pathogen in southern Africa. In order to determine the role of wild mammals in the natural ecology of the virus, sera from 3,772 wild mammals of 87 species and from 1,978 domestic dogs collected in South Africa and Zimbabwe between 1964 and 1985 were tested for antibody to CCHF virus by reversed passive hemagglutination inhibition (RPHI) and by indirect immunofluorescence (IF). Antibody was found to be highly prevalent in large mammals in the Orders Artiodactyla and Perissodactyla such as giraffe, Giraffa camelopardalis (3/3 positive), rhinoceros, Ceratotherium simium and Diceros bicornis (7/13), eland, Taurotragus oryx (59/127), buffalo, Syncerus caffer (56/287), kudu, Tragelaphus strepsiceros (17/78), and zebra, Equus burchelli (16/93). In small mammals antibody was found in the sera of 40/293 hares, 22/1,305 rodents, and 1/74 wild carnivores, but not in 522 primates, 176 insectivores, or 19 hyrax. Antibody was also found in the sera of 118/1,978 domestic dogs. The species of wild mammal in which antibody was distributed (with highest antibody prevalence in hares and large herbivores) reflects the feeding preference of immature and adult ticks of the genus Hyalomma, suggesting that Hyalomma sp. are the principal CCHF vectors in the wild.

Crimean-congo hemorrhagic fever in South Africa.

Crimean-Congo hemorrhagic fever virus was isolated for the first time in South Africa in February 1981, from the blood of a 13-year-old boy who died in Johannesburg after attending a camp in a nature reserve in the western Transvaal. Virus was isolated from 21/120 pools of questing ticks from the nature reserve, the infected species being Hyalomma marginatum rufipes and H. truncatum. Virus was also isolated from 4/38 pools of partially engorged ticks and other ectoparasites collected off hosts, the infected species being H.m. rufipes, H. truncatum and Rhipicephalus evertsi. Antibodies were found in the sera of 5/74 humans, 8/26 wild vertebrates, 74/270 sheep, and 109/170 cattle from the reserve and surrounding farms. Antibodies were also found in 28/200 hares from various locations in the country. It was concluded that the virus is widely prevalent in South Africa, but the full medical and veterinary significance of its presence has yet to be determined.

Epidemiologic and clinical features of Crimean-Congo hemorrhagic fever in southern Africa.

Following the diagnosis in 1981 of the first case of Crimean-Congo hemorrhagic fever (CCHF) in South Africa, an antibody survey was undertaken on cattle sera to determine the distribution of the virus and specific diagnostic tests were routinely applied to specimens from suspected cases of hemorrhagic fever to establish the medical significance of its presence. Antibody to CCHF virus was demonstrated by reversed passive hemagglutination-inhibition technique in 2,460/8,667 (28%) cattle sera and in 140/180 herds tested in South Africa, as well as in 347/763 (45%) cattle sera and in 32/34 (94%) herds tested in Zimbabwe. The antibody was found in all major cattle farming areas, but was of low prevalence along the southern coast where 2 of the 3 species of Hyalomma tick which occur in South Africa are absent. From February 1981 to January 1986, inclusive, 29 indigenous cases of CCHF were diagnosed in 16 outbreaks which arose in various locations throughout South Africa. A further 2 imported cases of CCHF arose in Zaire and Tanzania. The clinical features of infection conformed to the classical descriptions of CCHF in the Soviet Union. The fatal outcome in 11/31 cases indicates that the African disease is no less severe than that which occurs in Eurasia. It is inferred that the virus is widespread in all countries in Africa and Eurasia which lie within the limits of world distribution of ticks of the genus Hyalomma.

Comparison of electron microscopic techniques for enumeration of endogenous retrovirus in mouse and Chinese hamster cell lines used for production of biologics.

Murine myeloma and Chinese hamster ovary cells are used widely in the manufacture of recombinant proteins for biopharmaceuticals. However, rodent cell lines express endogenous retrovirus, which necessitates appropriate design of purification processes to remove virus in excess of the calculated maximum retroviral load. Currently, electron microscopy is the method of choice for determination of retroviral titre in bulk harvest. In this study we compared three electron microscopy techniques to determine retroviral titre in bulk harvest. These were direct negative stain, negative stain after sucrose-density purification and thin section electron microscopy of pelleted supernatant. The study demonstrated that the level of C-type retrovirus associated with cells was predictive of the viral load in cell culture supernatants. The most accurate method for quantifying viral load was direct counting, followed by thin section of pelleted supernatant and negative stain after sucrose concentration. The most practical method was thin section of resuspended pelleted supernatant, which gave improved detection limits.

High throughput detection of retrovirus-associated reverse transcriptase using an improved fluorescent product enhanced reverse transcriptase assay and its comparison to conventional detection methods.

The development and application of a novel, sensitive TaqMan fluorescent probe-based product enhanced RT test (F-PERT) for the detection of retrovirus are described. The assay allows discrimination between the amplification signals generated by genuine positive signals that result from retroviral RT activity and the RT-like activity from DNA polymerases. The RT-like activity from DNA polymerases was suppressed by the addition of activated calf-thymus DNA with no reduction in the RT activity. A linear relationship between threshold cycle (C(T)) and the number of virus particles was demonstrated, allowing quantification of retroviruses in unknown samples. The F-PERT assay was able to detect a wide range of retroviral RT activities, including that from porcine endogenous retrovirus (PoERV), murine leukaemia virus (MLV), simian foamy virus (SFV), simian immunodeficiency virus (SIVmac) and squirrel monkey retrovirus (SMRV). The detection limit of SMRV, MLV and PoERV was approximately 100 virion particles and the test was able to detect at least 10(2) molecules of purified RT enzyme. RT activity was not detected in cellular lysates and supernatants from MRC-5, BT, VERO, or Raji cells, whereas RT activity was detected in C1271, Mus dunni, K-Balb, BHK-21, CHO-K1, SP2/0-Ag14 and NSO cell supernatants. RT activity was also detected in the Spodoptera cell line Sf9.

Blood transfusion-associated Pseudomonas fluorescens septicaemia: is this an increasing problem?

Pseudomonas fluorescens transfusion-related septicaemia (TRS) is rare. We present the first description in the UK of two cases of TRS caused by this organism.

Plague in South African rodents 1972-1981.

Sera from 3012 rodents of 24 species captured in South Africa during the period 1972-81 were tested for antibody to the Fraction 1 antigen of Yersinia pestis by passive haemagglutination. Antibodies were found in seven (0.23%) rodents of three species. These were Desmodillus auricularis and Tatera brantsii in the northern Cape Province and Rhabdomys pumilio in the eastern Cape Province. Rodents were found positive in 1972, 1974, 1975 and 1979, indicating that plague continues to circulate in rodent populations apparently without causing human cases. The significance of the results is discussed in relation to plague outbreaks in neighbouring countries and to the 1982 outbreak in South Africa.