Automated differentiation of pre-diagnosis Huntington's disease from healthy control individuals based on quadratic discriminant analysis of the basal ganglia: the IMAGE-HD study.

We investigated two measures of neural integrity, T1-weighted volumetric measures and diffusion tensor imaging (DTI), and explored their combined potential to differentiate pre-diagnosis Huntington's disease (pre-HD) individuals from healthy controls. We applied quadratic discriminant analysis (QDA) to discriminate pre-HD individuals from controls and we utilised feature selection and dimension reduction to increase the robustness of the discrimination method. Thirty six symptomatic HD (symp-HD), 35 pre-HD, and 36 control individuals participated as part of the IMAGE-HD study and underwent T1-weighted MRI, and DTI using a Siemens 3 Tesla scanner. Volume and DTI measures [mean diffusivity (MD) and fractional anisotropy (FA)] were calculated for each group within five regions of interest (ROI; caudate, putamen, pallidum, accumbens and thalamus). QDA was then performed in a stepwise manner to differentiate pre-HD individuals from controls, based initially on unimodal analysis of motor or neurocognitive measures, or on volume, MD or FA measures from within the caudate, pallidum and putamen. We then tested for potential improvements to this model, by examining multi-modal MRI classifications (volume, FA and MD), and also included motor and neurocognitive measures, and additional brain regions (i.e., accumbens and thalamus). Volume, MD and FA differed across the three groups, with pre-HD characterised by significant volumetric reductions and increased FA within caudate, putamen and pallidum, relative to controls. The QDA results demonstrated that the differentiation of pre-HD from controls was highly accurate when both volumetric and diffusion data sets from basal ganglia (BG) regions were used. The highest discriminative accuracy however was achieved in a multi-modality approach and when including all available measures: motor and neurocognitive scores and multi-modal MRI measures from the BG, accumbens and thalamus. Our QDA findings provide evidence that combined multi-modal imaging measures can accurately classify individuals up to 15 years prior to onset when therapeutic intervention is likely to have maximal effects in slowing the trajectory of disease development.

The trouble with tribbles.

Cell division and cell movements must be coordinated during development. A novel inhibitor of cell division, Tribbles, has been identified that blocks mitosis at a critical point in Drosophila morphogenesis. The data support a role for Tribbles in promoting proteolytic degradation of String/Cdc25, a key regulator of mitosis.

Sodium butyrate inhibits myogenesis by interfering with the transcriptional activation function of MyoD and myogenin.

Sodium butyrate reversibly inhibits muscle differentiation and blocks the expression of many muscle-specific genes in both proliferating myoblasts and differentiated myotubes. We investigated the role of the basic helix-loop-helix (bHLH) myogenic determinator proteins MyoD and myogenin in this inhibition. Our data suggest that both MyoD and myogenin are not able to function as transcriptional activators in the presence of butyrate, although both apparently retain the ability to bind DNA. Transcription of MyoD itself is extinguished in butyrate-treated myoblasts and myotubes, an effect that may be due to the inability of MyoD to autoactivate its own transcription. We present evidence that the HLH region of MyoD is essential for butyrate inhibition of MyoD. In contrast to MyoD and myogenin, butyrate does not inhibit the ubiquitous basic HLH protein E2-5 from functioning as a transcriptional activator.

An ubiquitously expressed gene 3.5 kilobases upstream of the glycerol-3-phosphate dehydrogenase gene in mice.

While studying the organization of the mouse glycerol-phosphate dehydrogenase gene (Gdc-1 on chromosome 15), we identified a novel transcriptional unit located only 3.4 kilobases (kb) upstream of the 5' end of the Gdc-1 gene. This gene has been provisionally named D15Kz1. The unusual proximity of these two genes led us to investigate the pattern of expression and sequence characteristics of the new gene for comparison with those of Gdc-1. D15Kz1 was found to have transcripts of 3.2 and 3.4 kb in length. The 3.4-kb transcript was expressed at low levels in all tissues examined, whereas the 3.2-kb transcript was detected only in the cerebral cortex and the brown fat. D15Kz1 and Gdc-1 are not coordinately regulated, as evidenced by the characteristics of their expression in several tissues and in differentiating 3T3-F442A adipocyte cultures. A cDNA sequence of 3,105 bases isolated from an embryonal carcinoma lambda gt10 cDNA library had a large open reading frame of 461 amino acids at one end followed by 1.6 kb of sequence with multiple stop codons. Algorithms used to search the protein and nucleic acid data bases detected no significant sequence similarity to any other protein or gene. Southern blot analysis of genomic DNA using the D15Kz1 cDNA as a probe indicated that D15Kz1 is a single-copy gene in the mouse genome and that it is conserved in humans, rats, and chickens. This conservation of gene sequences suggests that D15Kz1 encodes a protein with an important cellular function.

Fidelity of two retroviral reverse transcriptases during DNA-dependent DNA synthesis in vitro.

We determined the fidelity of avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases (RTs) during DNA synthesis in vitro using the M13mp2 lacZ alpha gene as a mutational target. Both RTs commit an error approximately once for every 30,000 nucleotides polymerized. DNA sequence analysis of mutants generated in a forward mutation assay capable of detecting many types of errors demonstrated that avian myeloblastosis virus RT produced a variety of different mutations. The majority (58%) were single-base substitutions; all of which resulted from the misincorporation of either dAMP or dGMP. Minus-one frameshifts were also common, composing about 30% of the mutations. In addition to single-base events, eight mutants contained sequence changes involving from 2 to 59 bases. The frequency of these mutants suggests that, at least during DNA synthesis in vitro, RTs also commit errors by mechanisms other than classical base miscoding and misalignment. We examined the ability of RTs to synthesize DNA from a mismatched primer terminus at a sequence where the mismatched base was complementary to the next base in the template. Unlike cellular DNA polymerases which polymerize from the mismatched template-primer, RTs preferred to polymerize from a rearranged template-primer containing a matched terminal base pair and an unpaired base in the template strand. The unusual preference for this substrate suggests that the interactions between RTs and the template-primer are different from those of cellular DNA polymerases. The overall error rate of RT in vitro is sufficient to account for the estimated mutation rate of these viruses.

Improving the time efficiency of the Fourier synthesis method for slice selection in magnetic resonance imaging.

The design of slice selective pulses for magnetic resonance imaging can be cast as an optimal control problem. The Fourier synthesis method is an existing approach to solve these optimal control problems. In this method the gradient field as well as the excitation field are switched rapidly and their amplitudes are calculated based on a Fourier series expansion. Here, we provide a novel insight into the Fourier synthesis method via representing the Bloch equation in spherical coordinates. Based on the spherical Bloch equation, we propose an alternative sequence of pulses that can be used for slice selection which is more time efficient compared to the original method. Simulation results demonstrate that while the performance of both methods is approximately the same, the required time for the proposed sequence of pulses is half of the original sequence of pulses. Furthermore, the slice selectivity of both sequences of pulses changes with radio frequency field inhomogeneities in a similar way. We also introduce a measure, referred to as gradient complexity, to compare the performance of both sequences of pulses. This measure indicates that for a desired level of uniformity in the excited slice, the gradient complexity for the proposed sequence of pulses is less than the original sequence.

The homeobox gene cut interacts genetically with the homeotic genes proboscipedia and Antennapedia.

The cut locus (ct) codes for a homeodomain protein (Cut) and controls the identity of a subset of cells in the peripheral nervous system in Drosophila. During a screen to identify ct-interacting genes, we observed that flies containing a hypomorphic ct mutation and a heterozygous deletion of the Antennapedia complex exhibit a transformation of mouthparts into leg and antennal structures similar to that seen in homozygous proboscipedia (pb) mutants. The same phenotype is produced with all heterozygous pb alleles tested and is fully penetrant in two different ct mutant backgrounds. We show that this phenotype is accompanied by pronounced changes in the expression patterns of both ct and pb in labial discs. Furthermore, a significant proportion of ct mutant flies that are heterozygous for certain Antennapedia (Antp) alleles have thoracic defects that mimic loss-of-function Antp phenotypes, and ectopic expression of Cut in antennal discs results in ectopic Antp expression and a dominant Antp-like phenotype. Our results implicate ct in the regulation of expression and/or function of two homeotic genes and document a new role of ct in the control of segmental identity.

Retarded growth of Lucilia cuprina larvae on sheep and their sera following production of an immune response.

Attempts were made to immunize sheep against larvae of the sheep blowfly Lucilia cuprina using supernatant and pellet prepared by centrifuging (100,000 g max) homogenates from whole second instar larvae of L. cuprina or their excised guts. Injection of supernatant from whole larvae and from two fractions of this supernatant, prepared by ammonium sulphate precipitation, significantly reduced (by 24-58%) the final weight of larvae grown in vivo (i.e. on immunized sheep) for 20 or 44 h. Serum from animals vaccinated with supernatant from whole larvae reduced larval weights by 12% after growth for 20 h in vitro (i.e. on diet containing serum from treated animals). Pellet material from whole larvae or guts, when injected into sheep, stimulated an immune response which reduced the weight of larvae by 20-23% after 20 or 48 h in vitro. Larvae grown on these animals were not reduced in weight. Immunoglobulin (Ig) isolated from serum of a sheep vaccinated with gut material strongly retarded growth of larvae in vitro, the effect increasing with Ig concentration. These results indicate that an immune response in sheep, induced by injecting extracts of L. cuprina larvae, substantially reduces growth of this parasite.

Acquired resistance of sheep to larvae of Lucilia cuprina, assessed in vivo and in vitro.

The effect on subsequent larval survival of infesting sheep repeatedly with larvae of Lucilia cuprina was assayed in vivo and in vitro. One in vivo assay technique, in which implanted larvae were grown to third instar, indicated a significant reduction in larval survival; another in vivo technique, in which larvae were allowed to develop to second instar in small aluminium rings attached to the sheep, indicated no reduction in larval growth or survival. Larvae of Lucilia cuprina grown in vitro on media containing sera from previously infested sheep were significantly retarded in growth after 20 h compared with controls; no difference was detected when larvae were allowed to develop to pupation on two changes of the same media. No significant differences in survival of larvae either to 20 h or to pupation were obtained between the two treatments. ELISA antibody levels against crude soluble larval material were significantly higher for sera from infested sheep than for control sera, and the regression of antibody level on mean larval weight obtained after 20 h growth in vitro was significant. The immunoglobulin fraction isolated from sera of infested sheep significantly retarded larval growth when incorporated with normal serum in growth media. These results are consistent with an effect of specific anti-larval antibody produced by sheep in response to infestation.

Pattern- and growth-linked cell cycles in Drosophila development.

During Drosophila development the cell cycle is subject to diverse regulatory inputs. In embryos, cells divide in stereotypic patterns that correspond to the cell fate map. There is little cell growth during this period, and cell proliferation is regulated at G2/M transitions by patterned transcription of the Cdk1-activator, Cdc25/String. The string locus senses pattern information via a > 40 kb cis-regulatory region composed of many cell-type specific transcriptional enhancers. Later, in differentiated larval tissues, the cell cycle responds to nutrition via mechanisms that sense cellular growth. These larval cell cycles lack mitoses altogether, and are regulated at G/S transitions. Cells in developing imaginal discs exhibit a cycle that is regulated at both G1/S and G2/M transitions. G2/M progression in disc cells is regulated, as in the embryo, by string transcription and is thus influenced by the many transcription factors that interact with string's 'pattern-sensing' control region. G1/S progression in disc cells is controlled, at least in part, by factors that regulate cell growth such as Myc, Ras and phosphatidylinositol-3-kinase. Thus G1/S progression appears to be growth-coupled, much as in the larval endocycles. The dual control mechanism used by imaginal disc cells allows integration of diverse inputs which operate in both cell specification and cell metabolism.

Larval Schistosoma mansoni excretory-secretory glycoproteins (ESPs) bind to hemocytes of Biomphalaria glabrata (Gastropoda) via surface carbohydrate binding receptors.

Flow cytometric analysis of circulating blood cells (hemocytes) of Biomphalaria glabrata, molluscan intermediate host of Schistosoma mansoni, revealed the presence of 2 overlapping hemocyte subpopulations, designated R1 and R2. R1 hemocytes are characterized by their smaller size, reduced granularity, and the presence of the BGH1 surface epitope, whereas R2 cells are larger, more granulated, and generally lack the BGH1 cell marker. Both hemocyte subpopulations bound fluorescent dye (Oregon Green)-conjugated excretory-secretory glycoproteins (fESPs), although the specific fESP binding signal (geometric mean value), after correction for cellular autofluorescence, was greater in the R1 hemocyte subpopulation compared to that of the R2 subset. Partial inhibition of fESP binding to hemocytes consistently was achieved using various glycoconjugates (mucin, asialo-mucin, asialo-fetuin, heparin) and polysaccharides (fucoidan, dextran sulfate 8000), suggesting the involvement of hemocyte carbohydrate-binding receptors (CBRs) in reactions with ESP-associated glycans. Although sulfation of carbohydrate ligands contributed significantly to ESP blocking activity of some inhibitory polysaccharides and heparin, other sulfated proteoglycans (chondroitins A and B, heparan sulfate) were noninhibitory, indicating that charge alone was not solely responsible for the observed inhibition of hemocyte binding by fESPs. A similar blocking effect by desialylated glycoproteins (asialo-mucin, asialo-fetuin) further supports the contention that ESP-hemocyte interactions are mediated primarily through CBRs. The glycoconjugate inhibitors of ESP binding were only partially effective over a range of concentrations and their glycan moieties (oligosaccharides or long-chain polymers) comprised a diversity of major sugar groups, suggesting that hemocyte CBRs and S. mansoni larval ESPs likely represent a multiple receptor-ligand system. Previously reported findings of differential effects of ESPs on a variety of in vitro hemocyte functions are consistent with such a hypothesis.

Phenoloxidase activity in the reproductive system of Biomphalaria glabrata: role in egg production and effect of schistosome infection.

Infection by larval trematodes often causes a cessation of egg production in its molluscan intermediate host and is referred to as parasitic castration. Because phenoloxidase (PO) has been shown to be involved in egg formation in other invertebrate species, we investigated the role of PO in normal egg production in the snail, Biomphalaria glabrata, and the effects of Schistosoma mansoni infection on the PO pathway in this snail. Our data showed that PO activity in the albumen gland (AG) is initially expressed when snails reach a size of approximately 8 mm in shell diameter and continues to increase as snails grow, indicating a developmental link between snail size and AG PO expression. Egglaying was also shown to be coincidental with the onset of PO expression in the AG, thereby supporting a direct association between PO activity and egg production. In addition, exposure of snails to diethyldithiocarbamate (DDC), a PO inhibitor, affected normal in vivo egg production, as evidenced by a significant decrease in the numbers of eggs laid in DDC-treated groups compared to nontreated groups. Normal resumption of egg-laying activity in treated snails following withdrawal of the drug indicated that inhibition was reversible. Taken together, the results of our developmental and DDC-exposure studies provide strong support for a crucial role of PO in normal egg production in this animal. Finally, AG PO activities of infected and uninfected control snails were measured over the course of S. mansoni infection. Our results showed that both total and specific enzyme activities in the AG of infected snails were significantly decreased at 28 and 33 days postinfection (PI) when compared to those of control snails. Results of subsequent experiments assessing the effects of larval infection on L-tyrosine (PO substrate) levels in AG and ovotestis revealed a significant increase in the levels of this compound in both organs over the course of infection. It is concluded that AG PO activity is functionally linked to egg formation in normal snails and that a strong association exists between parasite-mediated decrease in AG PO activity and parasitic castration. However, from the data presented, a direct causal relationship linking infection, decreased PO, and castration has yet to be established.

Influence of culture medium and protein supplementation on in vitro oocyte maturation and fertilization in the domestic cat.

Domestic cat oocytes were cultured either in Waymouth MB 753/1 Medium (WAY) or in Eagle's Minimum Essential Medium (MEM) containing FSH, LH and estradiol-17beta and supplememted with one of the following: 5% fetal calf serum (FCS); 4 mg/ml bovine serum albumin (BSA); or 3 mg/ml polyvinylalcohol (PVA, a non-protein control). The oocytes were evaluated for: nuclear maturation after 48 hours of culture (in vitro maturation, IVM); fertilization and cleavage 24 to 30 hours postinsemination (in vitro fertilization, IVF); and early embryo development 48 hours postinsemination. Maturation rates were similar (P>0.05) for WAY + BSA (29.4%), MEM + BSA (46.7%) and MEM + PVA (43.3%), but were different (P<0.05) from the other treatments (range, WAY + FCS, 9.6% to WAY + PVA, 14.9%). Fertilization and cleavage rates were also similar (P>0.05) for WAY + BSA (51.4%, 30.5%), MEM + BSA (45.8%, 40.1%) and MEM + PVA (56.1%, 37.4%) and were greater (P<0.05) than all other treatments. These IVM/IVF oocytes were capable of culturing beyond 2-cells, with the highest proportion of 4- and 8- cell embryos forming in WAY and MEM media in the presence of BSA or in MEM medium containing PVA. In the domestic cat IVM/IVF system: both the type of culture medium and protein supplement influence the proportion of oocytes reaching Metaphase II; the type of protein supplement has a more significant (P<0.05) impact than medium on fertilization, cleavage and early embryo development; and nuclear maturation and fertilization in vitro can proceed in this species in the absence of supplementary protein.

Boophilus microplus: passage of bovine immunoglobulins and albumin across the gut of cattle ticks feeding on normal or vaccinated cattle.

Cattle were vaccinated with antigens from adult female Boophilus microplus and haemolymph was collected from female ticks which had engorged on these animals and on matched control cattle. Radio-immunoassay for bovine plasma proteins in haemolymph from ticks fed on control cattle showed low concentrations of IgG1 and albumin. There was a significant increase in bovine plasma proteins passing across the gut in ticks fed on vaccinated cattle, with an average of 150 times more albumin and four to five times more IgG1 in the haemolymph. Ticks with obviously damaged gut had the highest concentrations of bovine plasma proteins but apparently undamaged ticks from vaccinated cattle also had elevated protein concentrations.

The duration of latent infection and functional immunity in droughtmaster and hereford cattle following natural infection with Babesia argentina and Babesia bigemina.

Tne Droughtmaster and 9 Hereford cattle were born in an enzootic babesiasis area and became naturally infected with Babesia argentina and B.bigemina during a 3 year period. They were then kept free of cattle ticks (Boophilus microplus) for the remainder of the experiment. Annually for the next 3 years their individual infection status with Babesia was determined by sub-inoculation of blood into splenectomised calves. At the end of this period the functional immunity of all cattle was challenged by blood inoculation of heterologous strains of B. argentina and B. bigemina. Infection with B. argentina persisted in all Herefords for 2 years and in 7 for 3 years after they had been freed of B. microplus. The number of Droughtmasters with detectable B. argentina infection progressively declined, and at the end of 3 years only 2 of 10 were still infected. No Herefords were shown to be infected with B. bigemina following 1 year's freedom from B. microplus but latent B. bigemina infection of at least 2 year's duration was demonstrated in one of the Droughtmasters. A marked degree of resistance was apparent in all cattle when they were challenged with an heterologous strain of B. argentina. There were no differences between the response to challenge of the Herefords and Droughtmasters nor between the reactions of cattle which had apparently naturally sterilised B. argentina infection and those which were still infected. The heterologous strain of B. bigemina produced parasitaemia in the majority of animals but only minimal fever and anaemia resulted with no significant differences between the breeds.

The economics of cattle tick control in dry tropical Australia.

The economics of strategic dipping compared to nil treatment of cattle ticks (Boophilus microplus) on Droughtmaster cattle was assessed using a partial budget analysis. The analysis was based on reported experimental data which showed a bodyweight gain advantage from strategic dipping of 45 kg/head for growing cattle and 35 kg/head for breeding cows. Costs of dipping were calculated using 3 acaricide costs, that is 5.9, 20.9 and 62.7 per head and allowances were made for mustering, maintenance of facilities and annual cost of asset purchase under an intensive farm management system similar to the reported experimental conditions. The net gain of benefits over costs per annum for each acaricide cost was $927, $810 and $483 per 100 breeders and their progeny. Breakeven beef prices at which it was worth dipping were found to be 61, 69 and 94 per kg dressed weight depending on the cost of acaricide used for dipping. All prices and costs are expressed in 1981 dollars of purchasing power.

A comparison of direct fluorescent antibody and Giemsa staining for the post-mortem diagnosis of anaplasmosis.

Direct fluorescent antibody (DFA) and Giemsa staining of Anaplasma marginale were compared in smears collected serially at post-mortem (PM) from 11 experimentally infected calves. Once smears had been prepared and air-dried they could be held for at least 5 days before staining with either technique with no noticeable change in staining quality. DFA staining was more sensitive in detecting anaplasms in smears than Giemsa staining. Anaplasma spp could be differentiated from Babesia bovis and B. bigemina by DFA staining but there were cross reactions between A. marginale and A. centrale. Blood smears prepared from subcutaneous vessels in the legs provided better diagnostic material than kidney, heart and lung smears. Brain smears were not suitable for PM diagnosis using either staining technique.

An ancient defense system eliminates unfit cells from developing tissues during cell competition.

Developing tissues that contain mutant or compromised cells present risks to animal health. Accordingly, the appearance of a population of suboptimal cells in a tissue elicits cellular interactions that prevent their contribution to the adult. Here we report that this quality control process, cell competition, uses specific components of the evolutionarily ancient and conserved innate immune system to eliminate Drosophila cells perceived as unfit. We find that Toll-related receptors (TRRs) and the cytokine Spätzle (Spz) lead to NFκB-dependent apoptosis. Diverse "loser" cells require different TRRs and NFκB factors and activate distinct pro-death genes, implying that the particular response is stipulated by the competitive context. Our findings demonstrate a functional repurposing of components of TRRs and NFκB signaling modules in the surveillance of cell fitness during development.