Consequences of a screening programme on the prevalence of congenital hereditary sensorineural deafness in the Australian Cattle Dog.

Genetic disease testing programmes are used in domestic animal breeds to guide selective breeding with the aim of reducing disease prevalence. We assessed the change in the prevalence of canine congenital hereditary sensorineural deafness (CHSD) in litters of Australian Cattle Dogs following the introduction of a brainstem auditory evoked response (BAER) testing programme. We studied 608 pups from 122 litters from 10 breeding kennels. Despite 10 years of testing (1998-2008), no substantial reduction in prevalence of CHSD was evident in these 10 breeding kennels. Even for the subset of litters in which both parents were BAER tested as normal hearing (305 pups from 58 litters), there was no evidence of substantial reduction in prevalence. Odds ratios for CHSD in pups for each extra year since testing in the kennel commenced were 1.01 (95% CI, 0.88-1.17) and 1.03 (95% CI, 0.82-1.30) respectively for these populations. Amongst 284 dogs from 54 litters with extended pedigrees and both parents BAER-tested normal hearing, observed prevalences of CHSD were highest in pups with no BAER-tested normal grandparents (17% or 5/29) and lowest in pups with all four grandparents tested normal (0% or 0/9). In pups for which one, two and three grandparents tested negative, prevalences of CHSD were 12% (9/74), 9% (9/101) and 8% (6/71) respectively. Hence, testing programmes based on phenotypic screening may not lead to a substantial reduction in recessive genetic disease prevalence over the medium term, even when only tested normal parents are used. Exclusive breeding of litters in which both parents and all four grandparents are BAER-tested normal is expected to reduce CHSD prevalence in pups to the greatest extent over the long term.

Fast and Accurate Binary Response Mixed Model Analysis Via Expectation Propagation.

Expectation propagation is a general prescription for approximation of integrals in statistical inference problems. Its literature is mainly concerned with Bayesian inference scenarios. However, expectation propagation can also be used to approximate integrals arising in frequentist statistical inference. We focus on likelihood-based inference for binary response mixed models and show that fast and accurate quadrature-free inference can be realized for the probit link case with multivariate random effects and higher levels of nesting. The approach is supported by asymptotic calculations in which expectation propagation is seen to provide consistent estimation of the exact likelihood surface. Numerical studies reveal the availability of fast, highly accurate and scalable methodology for binary mixed model analysis. Supplementary materials for this article are available online.

Multiple interactions between SRm160 and SR family proteins in enhancer-dependent splicing and development of C. elegans.

BACKGROUND: SR family and SR-related proteins assemble on exonic splicing enhancer (ESE) sequences to promote both constitutive and regulated splicing. The SRm160 splicing coactivator, an SR-related nuclear matrix protein of 160 kDa, is important for the splicing of specific constitutive and ESE-dependent pre-mRNAs. RESULTS: In the present study, we show that SRm160 is required to promote pre-mRNA splicing mediated by a large population of functional ESE sequences within a randomized 18 nucleotide sequence. This suggests that it functions as a general coactivator by interacting with different SR family/SR-related proteins bound to different ESE sequences. Consistent with this, several SR family and SR-related proteins coimmunoprecipitated specifically with SRm160 in the presence of low salt. We used RNA interference (RNAi) in Caenorhabditis elegans to determine whether interactions between CeSRm160 and different CeSR family proteins are important in a whole-organism context. Previously we showed that RNAi of CeSRm160 and individual CeSR family genes other than CeSF2/ASF results in no obvious phenotype, which is indicative of gene redundancy. In the present study, we demonstrate that RNAi of CeSRm160 in combination with any CeSR family gene results in the production of unfertilized oocytes by the injected mother. CONCLUSIONS: The observation that simultaneous suppression of CeSRm160 and individual CeSR family proteins results in a distinct phenotype is indicative of critical functional interactions between these factors. Our results provide biochemical and genetic evidence indicating that interactions between SRm160 and multiple SR family proteins are important for both optimal splicing activity and for proper development.

Cuticle collagen genes. Expression in Caenorhabditis elegans.

Collagen is a structural protein used in the generation of a wide variety of animal extracellular matrices. The exoskeleton of the free-living nematode, Caenorhabditis elegans, is a complex collagen matrix that is tractable to genetic research. Mutations in individual cuticle collagen genes can cause exoskeletal defects that alter the shape of the animal. The complete sequence of the C. elegans genome indicates upwards of 150 distinct collagen genes that probably contribute to this structure. During the synthesis of this matrix, individual collagen genes are expressed in distinct temporal periods, which might facilitate the formation of specific interactions between distinct collagens.

cis regulatory requirements for hypodermal cell-specific expression of the Caenorhabditis elegans cuticle collagen gene dpy-7.

The Caenorhabditis elegans cuticle collagens are encoded by a multigene family of between 50 and 100 members and are the major component of the nematode cuticular exoskeleton. They are synthesized in the hypodermis prior to secretion and incorporation into the cuticle and exhibit complex patterns of spatial and temporal expression. We have investigated the cis regulatory requirements for tissue- and stage-specific expression of the cuticle collagen gene dpy-7 and have identified a compact regulatory element which is sufficient to specify hypodermal cell reporter gene expression. This element appears to be a true tissue-specific promoter element, since it encompasses the dpy-7 transcription initiation sites and functions in an orientation-dependent manner. We have also shown, by interspecies transformation experiments, that the dpy-7 cis regulatory elements are functionally conserved between C. elegans and C. briggsae, and comparative sequence analysis supports the importance of the regulatory sequence that we have identified by reporter gene analysis. All of our data suggest that the spatial expression of the dpy-7 cuticle collagen gene is established essentially by a small tissue-specific promoter element and does not require upstream activator or repressor elements. In addition, we have found the DPY-7 polypeptide is very highly conserved between the two species and that the C. briggsae polypeptide can function appropriately within the C. elegans cuticle. This finding suggests a remarkably high level of conservation of individual cuticle components, and their interactions, between these two nematode species.

Roy's largest root test under rank-one alternatives.

Roy's largest root is a common test statistic in multivariate analysis, statistical signal processing and allied fields. Despite its ubiquity, provision of accurate and tractable approximations to its distribution under the alternative has been a longstanding open problem. Assuming Gaussian observations and a rank-one alternative, or concentrated noncentrality, we derive simple yet accurate approximations for the most common low-dimensional settings. These include signal detection in noise, multiple response regression, multivariate analysis of variance and canonical correlation analysis. A small-noise perturbation approach, perhaps underused in statistics, leads to simple combinations of standard univariate distributions, such as central and noncentral [Formula: see text] and [Formula: see text]. Our results allow approximate power and sample size calculations for Roy's test for rank-one effects, which is precisely where it is most powerful.

Regulation of the Caenorhabditis elegans gut cysteine protease gene cpr-1: requirement for GATA motifs.

Expression of the Caenorhabditis elegans cysteine protease gene cpr-1 is regulated both spatially and temporally. In situ hybridisation and Northern blot analysis have shown that this gene is expressed exclusively in gut cells of all developmental stages except the embryo. We now show by transgenic transformation with cpr-1/lac Z reporter gene constructs that a sequence contained within the cpr-1 5' flanking region can direct this spatial and temporal expression. Deletion analysis of the cpr-1 promoter indicates that as little as 212 bp of upstream sequence is sufficient for this expression, although more upstream sequence may be involved in quantitative regulation of expression. Mutation of two GATA-like sequence elements at positions -51 and -147 upstream of the transcription start site ablates all expression, indicating an essential role in cpr-1 regulation. A concatemer of the cpr-1 -147 GATA motif placed upstream of minimal promoter/lac Z reporter gene constructs results in strong reporter gene expression in gut cells of larval stages and also in embryos. Weak expression is also detected in hypodermal cells. This pattern is reversed in the adult stage with strong expression in hypodermal cells and weaker expression in gut cells. Our findings suggest that spatial and temporal regulation of the cpr-1 gene is complex and involves activation by a GATA-like transcription factor.

Suspected fibrocartilaginous embolism in a cat.

A 12-year-old cat was presented to the University of Queensland's Small Animal Teaching Hospital with a 1-day history of left hemiparesis of acute onset, with no evidence of trauma or toxin exposure. Neurological examination findings were consistent with a lesion in the caudal left cervical spinal cord (C6 to C8), which was non-painful and had not progressed since the onset of clinical signs. No other abnormalities were found, although myelography showed a mild swelling involving the caudal cervical and cranial thoracic spinal segments. A diagnosis of suspected fibrocartilaginous embolism was made on the basis of the history, clinical presentation and diagnostic tests results, making this case the first report of a suspected fibrocartilaginous embolism in a cat that returned to normal function.

An autonomously replicating plasmid transforms Aspergillus nidulans at high frequency.

From an unstable Aspergillus nidulans colony, resulting from transformation with an A. nidulans gene bank, a plasmid was reisolated which transformed A. nidulans at a frequency of 20,000 transformants per 10(6) protoplasts at near saturation levels of transforming DNA. This represents a 250-fold enhancement of transformation efficiency over that found for typical integrative vectors such as pILJ16, the plasmid used in gene bank constructions. The plasmid, designated ARp1, is 11.5 kb in size, and consists of sequences derived from the 5.4-kb gene bank vector pILJ16, which carries the A. nidulans gene argB, and a 6.1-kb insert, designated AMA1. Southern analysis of transformant DNA showed ARp1 to be maintained in free form and not integrated into the chromosome. It has a mean copy number of 10-30 per haploid genome, and is mitotically unstable, being lost from 65% of asexual progeny of transformants. It shows similar transformational properties in A. niger and A. oryzae.

Isolation and characterisation of the crnA-niiA-niaD gene cluster for nitrate assimilation in Aspergillus nidulans.

Genomic clones containing the entire crnA-niiA-niaD gene cluster of Aspergillus nidulans have been isolated, and the structures of the niiA and niaD genes have been determined by nucleotide sequence analysis. This gene cluster is required for the assimilation of nitrate in A. nidulans, and the three genes encode a product required for nitrate uptake and the enzymes, nitrite reductase and nitrate reductase, respectively. The putative coding sequences, as deduced by comparison to cDNA clones of both niiA and niaD, are interrupted by multiple small introns, and the two genes are divergently transcribed. Identification and characterization of specific mRNAs involved in nitrate assimilation indicates that only monocistronic transcripts are involved, and that the approximate sizes of these transcripts are 1.6 kb, 3.4 kb and 2.8 kb for crnA, niiA and niaD, respectively. The results also indicate that control of niiA and niaD gene expression is mediated by the levels of mRNA accumulation, in response to the source of nitrogen in the growth medium. Two types of transcripts for niiA were observed.

Cuticular collagen genes from the parasitic nematode Ostertagia circumcincta.

The nematode cuticle is a multifunctional structure whose roles include exoskeleton and barrier between the animal and its environment. It is an extracellular matrix which consists predominantly of small collagen-like proteins. For those species studied, these cuticular collagens are encoded by a multigene family. In the free living nematode Caenorhabditis elegans, this family has approximately 100 members. Our data indicate a gene family of similar size in the parasitic nematode Ostertagia circumcincta. We have characterised a pair of tandemly duplicated collagen genes from O. circumcincta, colost-1 and colost-2, which we believe to be the direct homologues of col-12 and col-13, a tandemly duplicated pair previously identified in C. elegans. The interspecies comparison of these homologues indicates regions of extreme conservation. We conclude that the gene duplication event that resulted in the creation of col-12 and col-13 in C. elegans is most likely the same duplication that generated colost-1 and colost-2 in O. circumcincta, and thus this particular gene duplication precedes the divergence of the two species. These two nematode species are deeply diverged, O. circumcincta belonging to the order Strongylata and C. elegans to Rhabditata. The ability to identify direct homologues of individual cuticular collagen genes between deeply diverged species provides a powerful method for determining regions of structural importance in these small collagens. Characteristics that are conserved between homologues in divergent species, but not conserved with other members of the multigene family within one species, must relate to the specific function of that particular cuticular collagen.

Expression of Haemonchus contortus pepsinogen in Caenorhabditis elegans.

A genomic copy of a gut-expressed Haemonchus contortus candidate vaccine antigen, pepsinogen, was isolated using the polymerase chain reaction (PCR). The isolated sequence was 4 kb in length and contained eight introns ranging in size from 54 to 1475 base pairs. This sequence, together with its 3' non-coding DNA region containing a polyadenylation signal sequence, was cloned into the Bluescript SK(+) vector immediately downstream of the Caenorhabditis elegans cpr-5 gene promoter. This promoter has been shown previously to direct protein expression to the gut of C. elegans. The construct was micro-injected into DR96 unc-76(e911) mutant C. elegans together with a rescue plasmid and transgenic worms identified by reversion back to wild-type phenotype. Two transgenic lines of C. elegans were established. The presence of the injected construct and of the Haemonchus pepsinogen transcript in transgenic worms was confirmed by PCR analysis. Correct splicing of intronic sequences was observed. Immunohistochemistry showed expression of the Haemonchus pepsinogen protein in the gut of transgenic C. elegans, with reactivity evident in the larval and adult stages. Expression of the Haemonchus pepsinogen in C. elegans affirms the role of C. elegans as a model for parasitic nematodes and demonstrates its potential as a vector for expression of candidate vaccine antigens from parasitic nematodes.

Initial functional and economic status of patients with multivessel coronary artery disease randomized in the Bypass Angioplasty Revascularization Investigation (BARI).

Randomized trials of coronary angioplasty and bypass surgery have hypothesized that these procedures will have equivalent long-term rates of death and myocardial infarction. Functional status, quality of life, employment, and healthcare cost will therefore be critical measures of the efficacy of these alternative revascularization procedures. Patients at 7 sites in the Bypass Angioplasty Revascularization Investigation (BARI) were enrolled in an ancillary Study of Economics and Quality of Life (SEQOL). Physical function was assessed by the Duke Activity Status Index and emotional status by the Mental Health Inventory. Employment patterns and health care utilization were also measured at study entry and at 3-month intervals in follow-up. The 934 patients enrolled in SEQOL were similar to the 895 remaining BARI randomized patients. Most patients (63%) aged < or = 64 years were working, and almost all working patients (96%) intended to return to work. Patients aged > or = 65 years had lower household incomes but better health insurance coverage. Overall health ratings were significantly correlated with both physical and emotional status (p < 0.001). Patients enrolled in SEQOL are representative of the overall BARI population. Data collected in SEQOL will provide a detailed picture of the physical, emotional, and economic well-being after coronary angioplasty and bypass surgery.

Long-term cost-effectiveness of alternative management strategies for patients with life-threatening ventricular arrhythmias. Electrophysiologic Study versus Electrocardiographic Monitoring (ESVEM) Investigators.

BACKGROUND: Serial antiarrhythmic drug testing guided by Holter monitoring and electrophysiologic study had similar clinical outcomes in the Electrophysiologic Study versus Electrocardiographic Monitoring (ESVEM) trial, while patients treated with sotalol had improved outcomes. The purpose of this study was to compare long-term cost-effectiveness of these management alternatives. METHODS: Patients in the ESVEM trial were linked to computerized files of either the Health Care Finance Administration or the Department of Veterans Affairs. Total hospital costs and survival time over five year follow-up were measured using actuarial methods, and cost-effectiveness was calculated. RESULTS: Patients randomized to therapy guided by electrophysiologic study had more hospital admissions, higher costs, and a cost-effectiveness ratio of $162,500 per life year added compared with therapy guided by Holter monitoring. Patients randomized to sotalol had fewer hospitalizations, lower costs, and better survival than patients randomized to other drugs, and sotalol was a dominant strategy in the cost-effectiveness analysis. Patients for whom an effective drug was found had fewer hospital admissions, lower costs, and longer survival. These findings were robust in sensitivity analyses and in bootstrap replications. CONCLUSIONS: Serial drug testing guided by electrophysiologic study had an unfavorable cost-effectiveness ratio relative to Holter monitoring, while sotalol was cost-effective relative to other antiarrhythmic drugs.

Risk factors for the development of obliterative bronchiolitis after lung transplantation.

BACKGROUND: Acute rejection has emerged as an important risk factor for obliterative bronchiolitis after lung transplantation. We performed a multivariate analysis to assess the impact of additional variables. METHODS: Seventy-four recipients (48 heart-lung, 18 single-lung, and 8 bilateral-lung recipients) who survived longer than 90 days and underwent transplantation more than 15 months before data analysis were included in this study. Several variables were entered into a Cox regression analysis to determine their association with the development of bronchiolitis obliterans syndrome. RESULTS: Bronchiolitis obliterans syndrome developed in 48 (65%) of 74 patients. Significant correlations were detected for acute rejection score, defined as the sum of pathologic grades of each separate acute rejection episode (p = 0.0004, likelihood ratio test value = 12.4) and for lymphocytic bronchiolitis (p = 0.03). In a bivariate model, episodes of organizing pneumonia and bacterial or fungal pneumonia significantly increased the likelihood ratio test value of the acute rejection score. The addition of the cytomegalovirus infection score, reflecting the frequency and severity of infection, to the combination of the acute rejection score and episodes of bacterial or fungal pneumonia resulted in a further significant increase in the likelihood ratio test value. Significant risk factors for moderate to severe stages of airflow limitation were at least one episode of acute rejection of grade > or = 2, younger recipient age, and any acute rejection episode 180 days or longer after transplantation. CONCLUSIONS: Increasing frequency and severity of acute rejection episodes are strongly associated with the development of bronchiolitis obliterans syndrome. Lymphocytic bronchiolitis appeared to be significant by univariate analysis, but in a two-risk factor model, it did not augment the influence of acute rejection. Organizing pneumonia, bacterial or fungal pneumonia, and increasing severity and frequency of cytomegalovirus infections potentiate the effect of acute rejection. Late episodes of acute rejection and younger recipient age increase the risk for development of advanced disease.

Radiation-induced damage, repair and exchange formation in different chromosomes of human fibroblasts determined by fluorescence in situ hybridization.

We have used fluorescence in situ hybridization with whole-chromosome probes for human chromosomes 1, 4, 8 and 13 to investigate the extent to which the induction of damage and its repair after exposure to ionizing radiation is distributed randomly among these chromosomes. All the studies were performed with AG1522 human fibroblasts irradiated with 6 Gy and maintained in a nondividing state for at least 6 h after irradiation except for the measurements of initial damage. The extent of initial damage was determined by fusion of the cells immediately after irradiation with metaphase HeLa cells to obtain premature chromosome condensation (PCC). Breaks and exchanges were also scored by PCC 24 h after irradiation and in metaphase spreads at the first division after irradiation. The data obtained were consistent with random breakage and repair in these chromosomes. Comparing PCC 24 h after irradiation with first metaphase, there was a deficit in aberrations at metaphase, particularly in unrejoined breaks, implying loss or slowing of cells containing aberrations prior to the first division. An analysis of dicentrics and translocations in chromosome 4 at first and in subsequent divisions showed that there was an equal number of dicentrics and translocations at first metaphase with loss of dicentrics, but no loss of translocations in subsequent divisions. These data are supportive of the hypothesis tht the total number of chromosome aberrations in cells can be estimated from a single chromosome pair.

Isolation and characterization of four developmentally regulated cathepsin B-like cysteine protease genes from the nematode Caenorhabditis elegans.

Cathepsin B cysteine protease enzymes have been shown to be involved in a variety of different biological processes in eukaryotes. We have isolated and characterized four distinct cathepsin B-like genes from the genetically tractable nematode, Caenorhabditis elegans. This is the first reported finding of a cathepsin B-like multigene family within a nonparasitic metazoan. The four genes possess distinct genomic architectures, with variations in the position, number, and size of introns. The predicted amino acid sequences of the four genes are highly diverged. Phylogenetic analysis indicates the divergence of this multigene family within C. elegans is as great as the interspecies divergence between the vertebrates and nematode cathepsin B-like genes. In addition, each of the four genes described here shows a distinct temporal pattern of expression during C. elegans development.

Zidovudine susceptibility testing of human immunodeficiency virus type 1 (HIV) clinical isolates.

Traditional antiviral susceptibility testing methods using cell lines can be applied to no more than about 30% of clinical HIV isolates (Larder et al., 1989a; Fenyo et al., 1989). We tested the cell-free supernatant from low passage clinical HIV isolates using donor peripheral blood mononuclear cells (PBMC). Drug susceptibility was assessed by measuring the effect of increasing zidovudine (ZDV) concentrations on HIV P24 antigen production. Susceptibility results were obtained on 24/27 consecutive clinical isolates and 6/6 laboratory isolates. The mean IC90 of isolates from untreated patients was 0.008 microM ZDV (range: 0.002-0.038). The IC90s of isolates from ZDV-treated patients ranged from 0.007 to greater than 10 microM ZDV. All isolates with an IC90 < 0.1 microM ZDV had a wild type sequence at codon 215 of the HIV pol gene; 11/12 isolates with an IC90 > 0.1 microM ZDV had a mutation at codon 215 (P < 0.001). Among 16 ZDV-treated patients, there was a modest correlation between the change in CD4 count from the start of ZDV treatment and the IC90 of the patient's isolate following treatment (r = 0.51). Susceptibility testing using donor PBMC can be a sensitive means of testing a broad range of clinical HIV isolates.

Prostate-specific antigen doubling times in patients who have failed radical prostatectomy: correlation with histologic characteristics of the primary cancer.

OBJECTIVES: This study sought to characterize the postoperative prostate-specific antigen (PSA) doubling time and time to biochemical recurrence in patients who have failed radical prostatectomy. METHODS: Of 539 consecutive patients who underwent radical prostatectomy between 1984 and 1992, postoperative PSA levels in 80 initially became undetectable (less than 0.07 ng/mL) before eventually increasing, as evidenced by rising PSA levels above the residual cancer detection limit of the Tosoh AIA-600 immunoassay run in the ultrasensitive mode (i.e., 0.07 ng/mL or higher). The PSA doubling time and time to biochemical recurrence were calculated for each of the 80 patients and were correlated with the histopathologic variables from the operative specimen. RESULTS: Postoperative PSA doubling times were predicted by the extent of capsular penetration, percent Gleason grade 4 or 5, lymph node involvement, and tumor volume on univariate analysis and by capsular penetration, percent Gleason grade 4 or 5, lymph node involvement, and patient age on multivariate analysis. Times to recurrence were predicted by the presence of positive margins and percent Gleason grade 4 or 5 in both univariate and multivariate regression models. The PSA doubling time did not correlate with recurrence time. The median PSA doubling time for all patients was 284 days, and the median time to recurrence was 648 days. CONCLUSIONS: These results demonstrate that PSA doubling time and recurrence time are indicative of different biologic characteristics of recurrent prostate cancer: Doubling time appears to represent the aggressiveness of the original prostate cancer, whereas time to recurrence reflects the extent of residual postoperative disease. This information should aid in the selection of men who need greater vigilance during postoperative surveillance.

Serum prostate-specific antigen and the biologic progression of prostate cancer.

OBJECTIVES: We have previously shown that serum prostate-specific antigen (PSA) is proportional to prostate cancer volume and that progression of prostate cancer is proportional to volume, but other investigators have not found serum PSA to be as useful in predicting pathologic stage at the time of radical prostatectomy. Because our series is the only study to examine prospectively all radical specimens at 3-mm intervals, we have examined the relationship between serum PSA and the morphologic indicators of cancer progression in our first 350 radical prostatectomies. METHODS: Preoperative serum PSA level was tabulated in 350 consecutive patients with prostate adenocarcinoma and compared with morphologic variables in the radical prostatectomy specimen. Morphologic variables included cancer volume, histologic grade, capsular penetration, seminal vesicle invasion, and lymph node metastasis. RESULTS: Serum PSA showed strong correlation with all morphologic variables, which were highly intercorrelated. Serum PSA level was strongly correlated with cancer volume, histologic grade, and frequency of regional spread to lymph nodes. Close intercorrelations found between all variables were translated into a scale relating each level of serum PSA elevation to stage of disease in morphologic terms. Using this scale, serum PSA level can contribute to patient evaluation and treatment decisions in men with prostate cancer. CONCLUSIONS: Serum PSA is primarily determined by prostate cancer volume and secondarily by the percentage of high-grade cancer (Gleason grades 4 and 5) in the prostate. Because of this basic relationship, serum levels of PSA provide a clinically useful estimate of morphologic findings in the prostate. Serial PSA determinations should reflect the growth of the cancer as well as the gradual evolution of more malignant cells with the passage of time. The use of a serum PSA-based rating scale can contribute to patient evaluation and treatment decisions in men with prostate cancer.