Mitogenomic analysis of extant condor species provides insight into the molecular evolution of vultures.

The evolution of large vultures linked to mountainous habitats was accompanied by extreme physiological and behavioral specializations for energetically efficient flights. However, little is known on the genetic traits associated with the evolution of these obligate soaring scavengers. Mitochondrial DNA plays a vital role in regulating oxidative stress and energy production, and hence may be an important target of selection for flight performance. Herein, we characterized the first mitogenomes of the Andean and California condors, the world's heaviest flying birds and the only living representatives of the Vultur and Gymnogyps genus. We reconstructed the phylogenetic relationships and evaluated possible footprints of convergent evolution associated to the life-history traits and distributional range of vultures. Our phylogenomic analyses supported the independent evolution of vultures, with the origin of Cathartidae in the early Paleogene (~ 61 Mya), and estimated the radiation of extant condors during the late Miocene (~ 11 Mya). Selection analyses indicated that vultures exhibit signals of relaxation of purifying selection relative to other accipitrimorph raptors, possibly indicating the degeneration of flapping flight ability. Overall, our results suggest that the extreme specialization of vultures for efficient soaring flight has compensated the evolution of large body sizes mitigating the selection pressure on mtDNA.

NADPH oxidase 4 (Nox4) deletion accelerates liver regeneration in mice.

Liver is a unique organ in displaying a reparative and regenerative response after acute/chronic damage or partial hepatectomy, when all the cell types must proliferate to re-establish the liver mass. The NADPH oxidase NOX4 mediates Transforming Growth Factor-beta (TGF-ß) actions, including apoptosis in hepatocytes and activation of stellate cells to myofibroblasts. Aim of this work was to analyze the impact of NOX4 in liver regeneration by using two mouse models where Nox4 was deleted: 1) general deletion of Nox4 (NOX4-/-) and 2) hepatocyte-specific deletion of Nox4 (NOX4hepKO). Liver regeneration was analyzed after 2/3 partial hepatectomy (PH). Results indicated an earlier recovery of the liver-to-body weight ratio in both NOX4-/- and NOX4hepKO mice and an increased survival, when compared to corresponding WT mice. The regenerative hepatocellular fat accumulation and the parenchyma organization recovered faster in NOX4 deleted livers. Hepatocyte proliferation, analyzed by Ki67 and phospho-Histone3 immunohistochemistry, was accelerated and increased in NOX4 deleted mice, coincident with an earlier and increased Myc expression. Primary hepatocytes isolated from NOX4 deleted mice showed higher proliferative capacity and increased expression of Myc and different cyclins in response to serum. Transcriptomic analysis through RNA-seq revealed significant changes after PH in NOX4-/- mice and support a relevant role for Myc in a node of regulation of proliferation-related genes. Interestingly, RNA-seq also revealed changes in the expression of genes related to activation of the TGF-ß pathway. In fact, levels of active TGF-ß1, phosphorylation of Smads and levels of its target p21 were lower at 24 h in NOX4 deleted mice. Nox4 did not appear to be essential for the termination of liver regeneration in vivo, neither for the in vitro hepatocyte response to TGF-ß1 in terms of growth inhibition, which suggest its potential as therapeutic target to improve liver regeneration, without adverse effects.

Evaluation of C-reactive protein and its kinetics as a prognostic indicator in canine leptospirosis.

OBJECTIVE: To evaluate C-reactive protein at presentation and during hospitalisation in dogs with acute kidney injury resulting from leptospirosis to compare C-reactive protein at presentation in dogs with acute kidney injury of different aetiology and to study its correlation with markers of inflammation, azotaemia and survival. MATERIALS AND METHODS: Prospective observational study of 41 dogs with acute kidney injury secondary to leptospirosis and 15 control dogs with acute kidney injury of different aetiology. C-reactive protein was measured at presentation in both groups and daily for 7 days in a subgroup of 28 dogs with leptospirosis. The associations of C-reactive protein with neutrophil count, albumin, urea, creatinine and survival were analysed. RESULTS: C-reactive protein was increased at presentation in all dogs with leptospirosis but was not significantly different from dogs with acute kidney injury of different cause. It was associated with markers of inflammation (neutrophil count, albumin) but not with azotaemia (creatinine, urea). It decreased gradually from presentation to day 4, with significantly lower concentrations in survivors than non-survivors. Initial C-reactive protein was only weakly associated with outcome, but its average concentration from presentation to day 2 was more strongly associated. Absolute and relative changes in C-reactive protein during hospitalisation and creatinine at presentation were not associated with survival. CLINICAL SIGNIFICANCE: Serial assessment of C-reactive protein may improve outcome prediction in dogs with leptospirosis compared with a single measurement at presentation or with markers of renal function.

A DNA methylation map of human cancer at single base-pair resolution.

Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination.

Genome data from a sixteenth century pig illuminate modern breed relationships.

Ancient DNA (aDNA) provides direct evidence of historical events that have modeled the genome of modern individuals. In livestock, resolving the differences between the effects of initial domestication and of subsequent modern breeding is not straight forward without aDNA data. Here, we have obtained shotgun genome sequence data from a sixteenth century pig from Northeastern Spain (Montsoriu castle), the ancient pig was obtained from an extremely well-preserved and diverse assemblage. In addition, we provide the sequence of three new modern genomes from an Iberian pig, Spanish wild boar and a Guatemalan Creole pig. Comparison with both mitochondrial and autosomal genome data shows that the ancient pig is closely related to extant Iberian pigs and to European wild boar. Although the ancient sample was clearly domestic, admixture with wild boar also occurred, according to the D-statistics. The close relationship between Iberian, European wild boar and the ancient pig confirms that Asian introgression in modern Iberian pigs has not existed or has been negligible. In contrast, the Guatemalan Creole pig clusters apart from the Iberian pig genome, likely due to introgression from international breeds.

Whole-exome sequencing in splenic marginal zone lymphoma reveals mutations in genes involved in marginal zone differentiation.

Splenic marginal zone lymphoma (SMZL) is a B-cell neoplasm whose molecular pathogenesis remains fundamentally unexplained, requiring more precise diagnostic markers. Previous molecular studies have revealed 7q loss and mutations of nuclear factor κB (NF-κB), B-cell receptor (BCR) and Notch signalling genes. We performed whole-exome sequencing in a series of SMZL cases. Results confirmed that SMZL is an entity distinct from other low-grade B-cell lymphomas, and identified mutations in multiple genes involved in marginal zone development, and others involved in NF-κB, BCR, chromatin remodelling and the cytoskeleton.

The effect of dorsal raphe nucleus (DRN) lesions on the locomotor activity rhythm in mice.

The study employed electrical lesions of dorsal raphe nucleus (DRN) to determine the functional significance of those nuclei in the regulation of wheel-running activity rhythm in mice in light/dark (LD 12:12), constant light (LL), and constant dark (DD) conditions. The wheel-running records showed that raphe nucleus lesions resulted in few days' decrease in common activity and amplitude in LD. The activity phase was not compact but in fragmentary form, especially in DD condition. In some animals an earlier onset of activity after DRN lesion in LD was observed. In LL extension of the rhythm period occurred. Destruction of DRN only slightly modulates the wheel-running circadian rhythm in mice.

A highly specific and sensitive competitive enzyme-linked immunosorbent assay (ELISA) based on baculovirus expressed pseudorabies virus glycoprotein gE and gI complex.

A direct competition enzyme-linked immunosorbent assay (ELISA) based on baculovirus expressed complex of pseudorabies virus (PRV) glycoproteins E (gE) and I (gI) has been developed. For that purpose gE and gI genes of PRV were co-expressed in insect cells. Complex formation was confirmed by radioimmunoprecipitation assay. The specificity and sensitivity of the test were evaluated and compared with an ELISA using only gE as an antigen and a commercially available test. For validation, 245 negative sera and 165 positive sera have been tested. The gE/gI ELISA had a higher sensitivity and specificity when compared with the ELISA using only gE as the antigen. Both sensitivity and specificity were comparable with the commercially available test. Moreover, the test based on the baculovirus gE/gI complex allows the detection of anti-gE antibodies in pig serum as early as two weeks after infection. The gE/gI ELISA test is easy to perform; its additional advantage is that the gE/gI antigen can be produced in baculovirus system in large quantities without handling live pseudorabies virus.

One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses.

A one-tube reverse transcription-polymerase chain reaction (RT-PCR) for absolute feline coronavirus (FCoV) quantitation was developed. The assay is based on the 5' nuclease activity of the Thermus flavus (Tfl) polymerase and a fluorogenic probe which generates fluorescence when it is cleaved. The fluorogenic probe, also called TaqMan(TM) probe (Perkin Elmer, Foster City, USA), is an oligonucleotide designed to bind between the two PCR primers to the target cDNA and is labeled with a reporter and a quencher dye. In the intact probe, the quencher dye suppresses the fluorescence of the reporter dye by Forster-type energy transfer. During the polymerase extension steps the Tfl exonuclease activity cleaves the hybridised probe resulting in the generation of fluorescent emission of the reporter dye. The threshold cycle (C(T) value) indicates the increase of reporter fluorescence and is directly related to the initial amount of target cDNA or RNA, respectively. Fluorescence is monitored in real time after each cycle by a Perkin-Elmer ABI Prism 7700 Sequence Detector. After completion of amplification, the C(T) values of the samples are calculated back to a standard curve, generated by amplification of diluted standard molecules. The one-tube RT-PCR described below allows precise quantitation, is highly sensitive, rapid (no separate reverse transcription step and no post-amplification steps), easy to handle, allows for a high sample throughput, shows a very good reproducibility, and can be executed with a low risk of contamination. The design of the primers probe combination enables the detection of all known FCoV strains and is also useful for the detection of canine coronavirus, transmissible gastroenteritis virus and porcine respiratory coronavirus.

[Case presentation in small animal medicine. What is your diagnosis?]. / Fallvorstellung Kleintiermedizin. Welche Diagnose stellen Sie?

Atrial fibrillation and dilated cardiomyopathy were diagnosed in a 9-year old boxer who was examined because of anorexia and ascites. Longterm treatment included a diuretic (furosemide), an ACE-inhibitor (benazepril), digoxin, and a betablocker (atenolol). The prognosis for DCMP is unfavorable--the described dog died 11 months after diagnosis.

[5-t-7-year results of cementless Endler acetabular prosthesis]. / 5- bis 7-Jahresergebnisse der zementfreien Hüftgelenkspfanne nach Endler.

From September 1980 to March 1985, 123 cementless, polyethylene Endler acetabular sockets of the threaded, tapered type were implanted in 115 patients at the department of orthopaedic surgery, Balgrist, University of Zurich. The first 102 Endler sockets, in combination with a Zweymüller femoral prosthesis, have been followed up till April 1988, this corresponding to an average observation period of 57.4 months. 12.8% of the total number of qualitative criteria and the Sutherland method, and further 20.6% of the cases were suspected of having loosened, so that a figure of 33.3% (34 loosened sockets) resulted. A definite correlation between loosening and the following factors were determined: females, limping, Trendelenburg sign, period of implantation and severe, continuous pain. No correlation was found between either tolerable pain, age, weight, or para-articular ossification, and loosening. The cementless artificial hip joint of Endler/Zweymüller combination led to a definite improvement of independency and living quality through increase in mobility and reduction of pain.

Bile acid synthesis. Metabolism of 3 beta-hydroxy-5-cholenoic acid to chenodeoxycholic acid.

Metabolism of 3 beta-hydroxy-5-cholenoic acid to chenodeoxycholic acid has been found to occur in rabbits and humans, species that cannot 7 alpha-hydroxylate lithocholic acid. This novel pathway for chenodeoxycholic acid synthesis from 3 beta-hydroxy-5-cholenoic acid led to a reinvestigation of the pathway for chenodeoxycholic acid from 3 beta-hydroxy-5-cholenoic acid in the hamster. Simultaneous infusion of equimolar [1,2-3H]lithocholic acid and 3 beta-hydroxy-5-[14C]cholenoic acid indicated that the 14C enrichment of chenodeoxycholic acid was much greater than that of lithocholic acid. Thus, in all these species, a novel 7 alpha-hydroxylation pathway exists that prevents the deleterious biologic effects of 3 beta-hydroxy-5-cholenoic acid.

Competitive inhibition of cytochrome P-450scc by (22R)- and (22S)-22-aminocholesterol. Side-chain stereochemical requirements for C-22 amine coordination to the active-site heme.

Two diastereomeric aminocholesterols, (22R)-22-aminocholesterol and (22S)-22-aminocholesterol, are both found to be potent inhibitors of the biosynthesis of pregnenolone from cholesterol by purified bovine mitochondrial P-450scc. Both steroids are competitive versus cholesterol, but the stereochemically correct analog (22R)-22-aminocholesterol is bound approximately 1000 times more tightly than (22S)-22-aminocholesterol. The dissociation constants are 25 nM and 13 microM, respectively. Direct comparisons between spectroscopic and enzymatic properties of the two enzymesterol complexes and the 22-amino-23,24-bisnor-5-cholen-3 beta-ol complex are made, underlining the importance of the stereochemistry at the C-22 position.

Effect of P-450scc inhibitors on corticosterone production by rat adrenal cells.

Suspensions of rat adrenocortical cells produce corticosterone as the major glucocorticoid. Cholesterol side-chain cleavage, the initial and rate-limiting step in the glucocorticoid biosynthetic pathway, is catalyzed by P-450scc. We have examined the effect of a variety of P-450scc inhibitors on corticosterone production by isolated rat adrenocortical cells. These inhibitors include reversible, noncovalently interacting inhibitors as well as mechanism-based inhibitors which irreversibly inactivate P-450scc in vitro. (20S)-22-nor-22-thiacholesterol and (22R)-22-aminocholesterol cause 50% inhibition of corticosterone production at 4 microM and 30 nM, respectively. Inhibition by these compounds was essentially not time-dependent. (20R)-20-(1-hexynyl)-pregn-5-en-3 beta, 20-diol and (20R)-20-(1,5-hexdiynyl)-pregn-5-en-3 beta, 20-diol at 10 microM inhibited corticosterone production in a time-dependent manner, resulting in 30% inhibition of corticosterone production during a 100-min incubation. (20S)-20-(2-trimethylsilyl ethyl)-pregn-5-en-3 beta, 20-diol inhibited in a strongly time-dependent manner. At 10 microM this compound irreversibly inhibited more than 90% of the side-chain cleavage capacity of the cell during a 40-min incubation. Cells treated with this steroid did not regain their capacity for side-chain cleavage after removal of free steroid. None of the inhibitors described above inhibited production of corticosterone by cells supplied with pregnenolone, the product of the P-450scc reaction. We suggest that the only significant effect of these compounds under these conditions is inhibition of the side-chain cleavage enzyme.

Synthesis of 3 alpha,7 alpha-dihydroxy-5 beta-androstan-17-one.

A short and efficient method for the stereospecific synthesis of 3 alpha,7 alpha-dihydroxy-5 beta-androstan-17-one was accomplished from the readily available 4-androstene-3,17-dione. Key steps are the stereospecific and selective epoxidation of 4,6-androstadiene-3,17-dione, followed by hydrogenations with carefully selected reagents, solvents and reaction conditions.

Bile acid synthesis. Metabolism of 3 beta-hydroxy-5-cholenoic acid in the hamster.

Synthesis of 3 beta-hydroxy-5-[1,2-3H]cholenoic acid has permitted a study of its metabolism in bile-fistula hamsters that received the compound by intravenous infusion. Metabolites in bile were identified by reverse isotope dilution after their complete resolution by high pressure liquid chromatography using muPorasil. Recovery of administered radioactivity ranged from 21-60% in three animals. In each study, lithocholic acid (0.8-4.4%) and chenodeoxycholic acid (7.8-11.3%) were identified as metabolites of 3 beta-hydroxy-5-cholenoate and can be considered primary bile acids in the side-chain pathway of bile acid synthesis beginning with the oxidation of cholesterol to 26-hydroxycholesterol.