Comparative genomics of the T cell receptor µ locus in marsupials and monotremes.

T cells are a primary component of the vertebrate adaptive immune system. There are three mammalian T cell lineages based on their T cell receptors (TCR). The αß T cells and γδ T cells are ancient and found broadly in vertebrates. The more recently discovered γµ T cells are uniquely mammalian and only found in marsupials and monotremes. In this study, we compare the TCRµ locus (TRM) across the genomes of two marsupials, the gray short-tailed opossum and Tasmanian devil, and one monotreme, the platypus. These analyses revealed lineage-specific duplications, common to all non-eutherian mammals described. There is conserved synteny in the TRM loci of both marsupials but not in the monotreme. Our results are consistent with an ancestral cluster organization which was present in the last common mammalian ancestor which underwent lineage-specific duplications and divergence among the non-eutherian mammals.

Opossum Mammary Maturation as It Relates to Immune Cell Infiltration and Nutritional Gene Transcription.

The mammary gland has evolved to accommodate the developmental needs of offspring in species-specific ways. This is particularly true for marsupials. Marsupial milk content changes dramatically throughout lactation in ways appearing timed with neonatal ontogeny and behavior. Here we investigate morphological restructuring within the mammaries throughout lactation in the gray short-tailed opossum, Monodelphis domestica. Substantial remodeling of the mammaries occurs throughout the first half of active lactation. It is not until the latter half of lactation that opossum mammaries appear histologically similar to active eutherian mammaries. Noteworthy was the presence of eosinophils in early developing mammary tissue, which correlated with elevated abundance of transcripts encoding the chemokine IL-16. The presence and abundance of whey protein transcripts within the opossum mammaries were also quantified. Whey acidic protein (WAP) transcript abundance peaked in the latter half of lactation and remained elevated through weaning. Minimal transcripts for the marsupial-specific Early and Late Lactation Proteins (ELP/LLP) were detected during active lactation. Elevated abundance of LLP transcripts was only detected prior to parturition. Overall, the results support the role of eosinophils in mammary restructuring appearing early in mammalian evolution, and describe key similarities and differences in nutritional protein transcript abundance among marsupial species.

WITHDRAWN: Corrigendum to "The pathology and pathogenicity of a novel Haemoproteus spp. infection in wild Little Penguins (Eudyptula minor)" [Vet. Parasitol. 197 (1-2) (2013) 74-84].

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.vetpar.2014.07.021. The duplicate article has therefore been withdrawn.

The pathology and pathogenicity of a novel Haemoproteus spp. infection in wild Little Penguins (Eudyptula minor).

One hundred and thirty four Little Penguin (Eudyptula minor) carcases found since 2004 in south west Australia were necropsied. The livers and spleens from ten of the penguins exhibited varying degrees of multifocal, randomly scattered areas of necrosis and varying numbers of parasites were associated with these areas. Hepatomegaly and splenomegaly were noted in many of these ten cases. Necrosis and parasites were also observed in the cardiac muscle of four of the cases and in the lung tissue in one of the penguins. Using PCR, the parasites were positively identified in four of the cases as Haemoproteus spp. and morphologically identical tissue stage parasites associated with histopathological changes were observed in all ten dead penguins. This is the first study to demonstrate both the in situ presence of the Haemoproteus parasite in any member of the Sphensicidae family and mortality due to its presence. We postulate the involvement of anomalous environmental conditions in a potential increase in local vectors.

The epidemiology of published norovirus outbreaks: a review of risk factors associated with attack rate and genogroup.

The purpose of this study was to examine global epidemiological trends in human norovirus (NoV) outbreaks by transmission route and setting, and describe relationships between these characteristics, viral attack rates, and the occurrence of genogroup I (GI) or genogroup II (GII) strains in outbreaks. We analysed data from 902 reverse transcriptase-polymerase chain reaction-confirmed, human NoV outbreaks abstracted from a systematic review of articles published from 1993 to 2011 and indexed under the terms 'norovirus' and 'outbreak'. Multivariate regression analyses demonstrated that foodservice and winter outbreaks were significantly associated with higher attack rates. Foodborne and waterborne outbreaks were associated with multiple strains (GI+GII). Waterborne outbreaks were significantly associated with GI strains, while healthcare-related and winter outbreaks were associated with GII strains. These results identify important trends for epidemic NoV detection, prevention, and control.

A polymorphism in the VKORC1 regulator calumenin predicts higher warfarin dose requirements in African Americans.

Warfarin demonstrates a wide interindividual variability in response that is mediated partly by variants in cytochrome P450 2C9 (CYP2C9) and vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1). It is not known whether variants in calumenin (CALU) (vitamin K reductase regulator) have an influence on warfarin dose requirements. We resequenced CALU regions in a discovery cohort of dose outliers: patients with high (>90th percentile, n = 55) or low (<10th percentile, n = 53) warfarin dose requirements (after accounting for known genetic and nongenetic variables). One CALU variant, rs339097, was associated with high doses (P = 0.01). We validated this variant as a predictor of higher warfarin doses in two replication cohorts: (i) 496 patients of mixed ethnicity and (ii) 194 African-American patients. The G allele of rs339097 (the allele frequency was 0.14 in African Americans and 0.002 in Caucasians) was associated with the requirement for a 14.5% (SD +/- 7%) higher therapeutic dose (P = 0.03) in the first replication cohort and a higher-than-predicted dose in the second replication cohort (allele frequency 0.14, one-sided P = 0.03). CALU rs339097 A>G is associated with higher warfarin dose requirements, independent of known genetic and nongenetic predictors of warfarin dose in African Americans.

Clinical implications of sugammadex.

Sugammadex is a cyclodextrin molecule that encapsulates and inactivates rocuronium and vecuronium. As a result, any degree of neuromuscular block produced by rocuronium or vecuronium can be rapidly and completely reversed without autonomic effects. Because sugammadex is optimised for reversing rocuronium it is most likely to be used in conjunction with this drug. Sugammadex will allow deep levels of block to be maintained until the very end of surgery, and will allow block to be reversed at any time after rocuronium administration, even just a few minutes. The recommended dose-range is 2-16 mg.kg-1 (ascender), depending on the level of block. The availability of sugammadex reversal may increase the use of rocuronium, and decrease the use of suxamethonium and benzylisoquinoline neuromuscular blocking drugs. In addition, it will certainly increase pharmacy costs, which may be offset by faster recovery and discharge from the post-anesthesia recovery unit. Sugammadex may also change monitoring practices in that post-tetanic count will be required to quantify deep block, and quantitative monitoring of recovery may be driven by cost concerns in order to allow the use of the smallest dose of sugammadex that gives a satisfactory train-of-four ratio. Alternatively, monitoring may essentially be abandoned since a large dose of sugammadex will reliably reverse any degree of rocuronium-induced block. The ultimate clinical utility of sugammadex will be clear only after large-scale clinical use.

Effects of application in spring of urea fertiliser on aspects of reproductive performance of pasture-fed dairy cows.

AIMS: To assess if raising concentrations of crude protein (CP) in pasture in spring by the frequent application of urea fertiliser would affect ovarian follicular dynamics, luteal function, onset of oestrus and reproductive performance of dairy cows under farming conditions in New Zealand. METHODS: Spring-calved dairy cows were grazed for 101 days in paddocks that were either not fertilised (Control; n=20) during the course of the study, or were fertilised with 40-50 kg nitrogen (N)/ha every 4-6 weeks (High-N; n=20). Similar generous pasture allowances were offered to both groups. Concentrations of CP in pasture, urea in serum and progesterone in milk were measured. Ovarian follicular and luteal dynamics were determined using ultrasonography. Oestrous behaviour and the number, time and outcome of inseminations were also recorded. RESULTS: Mean concentrations of CP in pasture and urea in serum was higher in the High-N than the Control group (25.2 vs 21.6 and 8.3 vs 5.4 mmol/L for CP and urea, respectively; p<0.001). Intervals between calving and first oestrus, first insemination and conception, the time of first emergence of a dominant follicle, milk progesterone concentration, and the diameter of the corpus luteum (CL) in the first luteal phase did not differ significantly between groups. The interval from calving to first ovulation tended (p=0.10) to be lower and the diameter of the dominant follicle of the oestrous cycle at which cows conceived was greater (p=0.02) in Control than High-N cows. CONCLUSIONS: The use of large amounts of urea fertiliser during spring and the consequent increases in concentrations of CP in pasture and urea in serum did not negatively affect any of the parameters of reproductive performance of pasture-fed dairy cows that were assessed in this study.

Patients' perceived health status following primary surgery for oral and oropharyngeal cancer.

How oral and oropharyngeal cancer patients view their 'quality of life' is of fundamental importance. Any differences seen in their health state compared with normative data and with other disease conditions allows a wider perspective on their outcome after surgery. A cross-sectional postal survey was undertaken of patients treated for oral/oropharyngeal squamous cell carcinoma by primary surgery using the University of Washington Quality of Life Questionnaire Version 4 (UW-QOL v4) and the EuroQol EQ-5D. Of 348 patients surveyed, 224 returned analysable forms, (response rate 64%). In the EQ-5D items, 40% of the group reported a problem in walking, 23% with self-care, 44% in performing usual activities, 50% with pain or discomfort and 33% with anxiety or depression. The mean overall health visual analogue scale (VAS) score was 74 (SE 1) minimum 30 and maximum 100. The mean utility (health index) score was 0.75 (SE 0.02) minimum -0.18 and maximum 1.0. Compared to national reference data, patients in our cohort of under 60 years of age fared significantly worse than expected for their age but this was not so for older patients. There were strong correlations between appropriate domains of the EQ-5D and UW-QOLv4 and between UW-QOL global measures and EQ-5D VAS.

Linkage mapping and physical localization of the major histocompatibility complex region of the marsupial Monodelphis domestica.

We used genetic linkage mapping and fluorescence in situ hybridization (FISH) to conduct the first analysis of genic organization and chromosome localization of the major histocompatibility complex (MHC) of a marsupial, the gray, short-tailed opossum Monodelphis domestica. Family based linkage analyses of two M. domestica MHC Class I genes (UA1, UG) and three MHC Class II genes (DAB, DMA, and DMB) revealed that these genes were tightly linked and positioned in the central region of linkage group 3 (LG3). This cluster of MHC genes was physically mapped to the centromeric region of chromosome 2q by FISH using a BAC clone containing the UA1 gene. An interesting finding from the linkage analyses is that sex-specific recombination rates were virtually identical within the MHC region. This stands in stark contrast to the genome-wide situation, wherein males exhibit approximately twice as much recombination as females, and could have evolutionary implications for maintaining equality between males and females in the ability to generate haplotype diversity in this region. These analyses also showed that three non-MHC genes that flank the MHC region on human chromosome 6, myelin oligodendrocyte glycoprotein (MOG), bone morphogenetic protein 6 (BMP6), and prolactin (PRL), are split among two separate linkage groups (chromosomes) in M. domestica. Comparative analysis with eight other vertebrate species suggests strong conservation of the BMP6-PRL synteny among birds and mammals, although the BMP6-PRL-MHC-ME1 synteny is not conserved.

Linkage disequilibrium maps constructed with common SNPs are useful for first-pass disease association screens.

To develop an efficient strategy for mapping genetic factors associated with common diseases, we constructed linkage disequilibrium (LD) maps of human chromosomes 5, 7, 17, and X. These maps consist of common single nucleotide polymorphisms at an average intermarker distance of 100 kb. The genotype data from these markers in a panel of American samples of European descent were analyzed to produce blocks of markers in strong pair-wise LD. Power calculations were used to guide block definitions and predicted that high-level LD maps would be useful in initial genome scans for susceptibility alleles in case-control association studies of complex diseases. As anticipated, LD blocks on the X chromosome were larger and covered more of the chromosome than those found on the autosomes.

Molecular cloning of four lambda light chain cDNAs from the Australian brushtail possum Trichosurus vulpecula.

A brushtail possum mesenteric lymph node cDNA library was screened with a grey short-tail opossum Clambda probe and four immunoglobulin lambda cDNAs were isolated. Two of the isolated clones (L5 and L10) contained identical framework 4 regions and constant regions (but different variable regions), suggesting that the possum lambda locus is organized as multiple J-C pairs--a feature seen in the opossum and placental mammals. The cloning of the lambda light chain cDNAs signifies the completion of the basic molecular characterization of the brushtail possum immunoglobulin repertoire. The availability of this sequence data will allow extensive analysis of the immune response of the brushtail possum at the molecular level, as well as the development of specific immunological reagents for detection of immunoglobulin molecules at the protein level.

Characterization of Chlamydia pneumoniae persistence in HEp-2 cells treated with gamma interferon.

Infection with Chlamydia pneumoniae has been implicated as a potential risk factor for atherosclerosis. This study demonstrated the effects of gamma interferon (IFN-gamma)-mediated indoleamine 2,3-dioxygenase activity on C. pneumoniae persistence in HEp-2 cells, inclusion morphology, and ultrastructure. C. pneumoniae replication showed a dose-dependent decrease when treated with increasing concentrations of IFN-gamma and a phenotypic switch resulting in a decrease in typical inclusions with an increase in smaller, less-dense atypical inclusions. Ultrastructural analysis of IFN-gamma-treated C. pneumoniae revealed atypical inclusions containing large reticulatate-like aberrant bodies with no evidence of redifferentiation into elementary bodies.

The birth and death of human single-nucleotide polymorphisms: new experimental evidence and implications for human history and medicine.

Extensive, new databases of single-nucleotide polymorphisms (SNPs) provide a powerful resource for disease gene discovery, and they will be even more useful as more frequency data become available. Interesting observed genomic patterns include SNP deserts (regions of low SNP incidence) and lengthy regions of linkage disequilibrium containing only a few haplotypes. A variety of genetic studies will benefit from SNP resources.

Massive suprachoroidal hemorrhage during pars plana vitrectomy associated with Valsalva maneuver.

PURPOSE: To report the intraoperative occurrence of massive intraocular suprachoroidal hemorrhage associated with Valsalva maneuver. METHODS: Retrospective, multicenter study of patients who developed massive choroidal hemorrhage associated with Valsalva maneuver during vitrectomy. RESULTS: Massive intraoperative suprachoroidal hemorrhage in seven patients (seven eyes) involved three men and four women with a median age of 52 years (range, 26 to 82 years). General anesthesia was used in six of seven cases. Coughing or "bucking" on the endotracheal tube during general anesthesia or severe coughing during the one vitrectomy performed under local anesthesia was associated with massive suprachoroidal hemorrhage. In five of seven eyes, this occurred near the end of surgery, after air-fluid exchange but before sclerotomy closure. Scleral plugs were immediately placed, and sclerotomy closure was performed exigently. Immediate posterior sclerotomy was performed on five of seven eyes; an additional patient underwent posterior sclerotomy postoperatively. After median follow-up of 18 months (range, 3 to 36 months), final visual acuity was no light perception in four eyes, light perception in one eye, 20/250 in one eye, and 20/20 in one eye. Four eyes became phthisical. CONCLUSIONS: Valsalva maneuver during pars plana vitrectomy may result in massive suprachoroidal hemorrhage with disastrous visual consequences. Precautionary measures to prevent coughing or "bucking" on the endotracheal tube during general anesthesia, or a prolonged episode of coughing during local anesthesia, may prevent this potentially devastating complication.

Universal SNP genotyping assay with fluorescence polarization detection.

The degree of fluorescence polarization (FP) of a fluorescent molecule is a reflection of its molecular weight (Mr). FP is therefore a useful detection methodfor homogeneous assays in which the starting reagents and products differ significantly in Mr. We have previously shown that FP is a good detection method for the single-base extension and the 5'-nuclease assays. In this report, we describe a universal, optimized single-base extension assay for genotyping single nucleotide polymorphisms (SNPs). This assay, which we named the template-directed dye-terminator incorporation assay with fluorescence polarization detection (FP-TDI), uses four spectrally distinct dye terminators to achieve universal assay conditions. Even without optimization, approximately 70% of all SNP markers tested yielded robust assays. The addition of an E. coli ssDNA-binding protein just before the FP reading significantly increased FP values of the products and brought the success rate of FP-TDI assays up to 90%. Increasing the amount of dye terminators and reducing the number of thermal cycles in the single-base extension step of the assay increased the separation of the FP values benveen the products corresponding to different genotypes and improved the success rate of the assay to 100%. In this study the genomic DNA samples of 90 individuals were typed for a total of 38 FP-TDI assays (using both the sense and antisense TDI primers for 19 SNP markers). With the previously described modifications, the FP-TDI assay gave unambiguous genotyping data for all the samples tested in the 38 FP-TDI assays. When the genotypes determined by the FP-TDI and 5'-nuclease assays were compared, they were in 100% concordance for all experiments (a total of 3420 genotypes). The four-dye-terminator master mixture described here can be used for assaying any SNP marker and greatly simplifies the SNP genotyping assay design.

Nigrostriatal collaterals to thalamus degenerate in parkinsonian animal models.

Movement, cognition, emotion, and positive reinforcement are influenced by mesostriatal, mesocortical, and mesolimbic dopamine systems. We describe a fourth major pathway originating from mesencephalic dopamine neurons: a mesothalamic system. The dopamine transporter, specific to dopamine containing axons, was histochemically visualized in thalamic motor and limbic-related nuclei and regions that modulate behavioral state as opposed to sensory nuclei in rats, nonhuman primates, and humans. Anatomical tracing established this innervation's origin via axon collaterals from the mesostriatal pathway. These findings implicate the thalamus as a novel site for disease specific alterations in dopamine neurotransmission, such as exist with nigral degeneration attending Parkinson's disease. This was confirmed in hemiparkinsonian animals where reduction of thalamic dopamine innervation occurred coincident with signs of active axonal degeneration. Individual mesencephalic dopamine neurons therefore have the potential to modulate normal and pathologic behavior not only through traditional nigrostriatal pathways but also by way of axon collaterals that innervate the thalamus.