Immunohistochemical analysis of expression of VEGFR2, KIT,PDGFR-β, and CDK4 in canine urothelial carcinoma

Urothelial carcinomas (UCs), also known as transitional cell carcinomas, are the most common canine urinary tract neoplasms. Tyrosine kinases (TKs) are enzymes that tightly regulate cell growth and differentiation through phosphorylation. Receptor TK (RTK) inhibitors are currently used to treat UCs. Toceranib phosphate (Palladia; Pfizer) is an RTK inhibitor that blocks the activity of vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor–alpha and –beta (PDGFR-α, -β), FMS-like tyrosine kinase 3, stem cell factor receptor (KIT, kinase inhibitor targeting), and colony stimulating factor receptor. To better understand UCs and validate treatment targets, we performed immunohistochemical staining for RTKs, as well as a novel target, cyclin-dependent kinase 4 (CDK4, a central regulator of the mammalian cell cycle), on formalin-fixed, paraffin-embedded tissues from bladder biopsies from 17 dogs with UCs, 17 dogs with cystitis (diseased controls), and 8 normal dogs (negative controls). Although immunohistochemical scores could not be extrapolated to prognostic value, response to treatment, and outcome of patients with UC, we demonstrated expression of PDGFR-β and VEGFR2 in UCs; all UC samples staining positively for VEGFR2. Minimal positive staining for KIT was noted in the tumor samples. CDK4 staining intensity was significantly weaker in UCs compared with normal and cystitis bladder samples. The intense staining of VEGFR2 in UC cells suggested that VEGFR2 may be of prognostic and/or therapeutic value in dogs with UC. Overexpression of VEGFR2 in UC cells validates this receptor as a treatment target in UC.     

Lifelong population connectivity between large rivers and their tributaries: A case study of shovelnose sturgeon from the Mississippi and Wisconsin rivers

Fishes that depend on large‐river habitats have declined in many regions due to human alteration of both mainstems and tributaries. Conserving these species is often complicated by their dependence on long‐distance movements to complete their life cycle, but the location of key habitats is difficult to assess across multiple life history stages. Movement of large‐river fishes between mainstems and major tributaries is commonly observed, but the role of these exchanges at the population level is uncertain. We used shovelnose sturgeon (Scaphirhynchus platorynchus) as a case study for assessing the lifelong spatial ecology of large‐river fishes. Fin ray microchemistry data indicate that shovelnose sturgeon from the Mississippi and Wisconsin rivers represent a unified population that relies on connections among multiple large‐river habitats throughout life history. The tributary serves as an important spawning area, but fin ray chemistry data suggest larvae subsequently drift downstream to nursery habitats in the mainstem; an assertion that is supported by extensive field survey data. Our analyses underscore the lifelong importance of connections within river networks for maintaining viable populations of this, and likely other large‐river species that have been documented to use tributary habitats. These findings support a metapopulation perspective on species that depend on large‐river habitats, many of which are now highly altered and fragmented. For shovelnose sturgeon and species with similar life history, protecting or restoring linkages between mainstem rivers and their tributaries should be a conservation priority.     

Sediment geochemistry of streams draining abandoned lead/zinc mines in central Wales: the Afon Twymyn

Purpose  

Despite the decline of metal mining in the UK during the early 20th century, a substantial legacy of heavy metal contamination persists in river channel and floodplain sediments. Poor sediment quality is likely to impede the achievement of ‘good’ chemical and ecological status for surface waters under the European Union Water Framework Directive. This paper examines the environmental legacy of the Dylife lead/zinc mine in the central Wales mining district. Leachable heavy metal concentrations in the bed sediments of the Afon Twymyn are established and the geochemical partitioning, potential mobility and bioavailability of sediment-associated heavy metals are established.     

Long-term effects of fragmentation and fragment properties on bird species richness in Hawaiian forests

Forest fragmentation is a common disturbance affecting biological diversity, yet the impacts of fragmentation on many forest processes remain poorly understood. Forest restoration is likely to be more successful when it proceeds with an understanding of how native and exotic vertebrates utilize forest patches of different size. We used a system of forest fragments isolated by volcanic activity 153 years ago in Hawaii to examine how long-term fragmentation, as well as fragment size and structural features affect the richness of native and exotic bird species. The total number of bird species increased rapidly with forest fragment size, with most of the native species pool found in patches <3 ha. Smaller fragments were dominated by native bird species with several exotic bird species found only in the largest fragments, suggesting that exotic bird species in this landscape show greater area-sensitivity than native species. We used airborne scanning light detection and ranging (LiDAR) to assess whether fragment area was correlated with estimates of fragment vegetation volume as well as measures of tree height. Fragment area was highly correlated with vegetation volume, maximum tree height, and canopy height heterogeneity, and these variables were strong predictors of bird richness, demonstrating that remote sensing can provide key insights into the relationship between fragment structural attributes and biodiversity indicators. Overall, this work demonstrates the value of conserving small remnant mid-elevation forest patches for native birds in Hawaii. This work also provides insight into how newly created forest patches might be used by native and exotic bird species in Hawaii.     

Investigations on the effects of growth rate and dietary vitamin C on skeletal muscle collagen and hydroxylysyl pyridinoline cross-link concentration in farmed Atlantic salmon (Salmo salar)

We have investigated the interactions between dietary vitamin C levels (at 33, 79, 135, and 424 mg kg-1 of wet mass feed) and growth rate on the collagen and cross-link contents of fast muscle in farmed juvenile Atlantic salmon (Salmo salar L.). The growth rate was measured over an 11 week period using the thermal growth coefficient (TGC). Alkaline-soluble (0.1 M NaOH) (a-s) hydroxyproline (HYP) and alkaline-insoluble (i-s) HYP were determined as a measure of collagen content and hydroxylysyl pyridinoline (PYD) as a measure of mature collagen cross-link concentration. There was a approximately 5-fold increase in muscle vitamin C concentration at similar feed conversion ratios ( approximately 0.82) as dietary vitamin C levels increased from 39 to 424 mg kg-1 of wet mass feed. However, even the lowest dietary vitamin C was sufficient for normal skeletal development and growth. The lowest dietary vitamin C level tested resulted in a approximately 27% decrease in the a-sHYP concentration relative to the other diets, whereas there was no significant effect of vitamin C on the i-sHYP and PYD concentrations. ANOVA revealed no significant interaction between vitamin C and growth rate, whereas the covariate TGC was significant for i-sHYP and PYD but not for a-sHYP. Pyridinoline cross-link and i-s HYP concentrations were 11.1 and 7.7% lower, respectively, in high (TGC > 3.9) mass than low (TGC < 3.9) growth rate fish. These small differences in collagen cross-linking were associated with a 15.6% decrease in fillet firmness measured with an instrumental texture analyzer. It was concluded that for healthy juvenile salmon reared under controlled growth conditions, the dietary vitamin C inclusion of 79 mg kg-1 of wet mass feed was sufficient to produce the required synthesis of soluble muscle collagen. Furthermore, post-translational modifications of the collagen leading to cross-linking showed a small decrease with increasing growth rate but was independent of vitamin C concentration in the diet at these levels.     

Aggregation of rennet-altered casein micelles at low temperatures

The rennet-induced coagulation of bovine milk at 10 degrees C was investigated. The rate of change of absorbance at 600 nm was higher in milk renneted at 30 degrees C than that at 10 degrees C. The amount of casein sedimented on centrifuging skim milk at 5000g for 1 h at 10 degrees C increased with time after renneting. The viscosity of milk at 10 degrees C at low shear rates did not change significantly until 10 h after rennet addition, but it increased markedly after 20 h. Smaller particles in milk at 10 degrees C disappeared slowly over 36 h after rennet addition and aggregated into larger particles. These results suggested that casein micelles in milk aggregate at low temperatures. Reasons for the slow aggregation of milk renneted at 10 degrees C were investigated by inhibiting chymosin activity by pepstatin A. It is likely that beta-casein, or its hydrolysis, plays a role in aggregation of rennet-altered casein micelles at low temperatures.     

Carotenoid biosynthetic pathway in the citrus genus: number of copies and phylogenetic diversity of seven genes

The first objective of this paper was to analyze the potential role of allelic variability of carotenoid biosynthetic genes in the interspecific diversity in carotenoid composition of Citrus juices. The second objective was to determine the number of copies for each of these genes. Seven carotenoid biosynthetic genes were analyzed using restriction fragment length polymorphism (RFLP) and simple sequence repeats (SSR) markers. RFLP analyses were performed with the genomic DNA obtained from 25 Citrus genotypes using several restriction enzymes. cDNA fragments of Psy, Pds, Zds, Lcy-b, Lcy-e, Hy-b, and Zep genes labeled with [alpha-(32)P]dCTP were used as probes. For SSR analyses, two primer pairs amplifying two SSR sequences identified from expressed sequence tags (ESTs) of Lcy-b and Hy-b genes were designed. The number of copies of the seven genes ranged from one for Lcy-b to three for Zds. The genetic diversity revealed by RFLP and SSR profiles was in agreement with the genetic diversity obtained from neutral molecular markers. Genetic interpretation of RFLP and SSR profiles of four genes (Psy1, Pds1, Lcy-b, and Lcy-e1) enabled us to make inferences on the phylogenetic origin of alleles for the major commercial citrus species. Moreover, the results of our analyses suggest that the allelic diversity observed at the locus of both of lycopene cyclase genes, Lcy-b and Lcy-e1, is associated with interspecific diversity in carotenoid accumulation in Citrus. The interspecific differences in carotenoid contents previously reported to be associated with other key steps catalyzed by PSY, HY-b, and ZEP were not linked to specific alleles at the corresponding loci.     

Factors affecting the retention of rennet in cheese curd

The coagulant retained in cheese curd is a major contributor to proteolysis during ripening. The objective of this study was to quantify the effects of several milk-related factors and parameters during cheese manufacture on the retention of coagulant in cheese curd. The amount of coagulant retained in curd was determined by its activity on a synthetic heptapeptide (Pro-Thr-Glu-Phe-[NO2-Phe]-Arg-Leu) using reversed-phase HPLC. The retention of chymosin in cheese curd increased significantly when the pH of milk was reduced at rennet addition below pH 6.1, the pH at whey drainage below pH 5.7, or the average casein micelle size in milk and when the ionic strength of milk was increased. The casein content of milk and the quantity of chymosin added to milk had no significant effect on the retention of chymosin in curd; the quantity of coagulant bound per gram of casein remained unchanged.     

Tissue Doppler imaging for detection of radial and longitudinal myocardial dysfunction in a family of cats affected by dystrophin-deficient hypertrophic muscular dystrophy

Diagnosis of feline hypertrophic cardiomyopathy currently is based on the presence of myocardial hypertrophy detected using conventional echocardiography. The accuracy of tissue Doppler imaging (TDI) for earlier detection of the disease has never been described. The objective of this sudy was to quantify left ventricular free wall (LVFW) velocities in cats with hypertrophic muscular dystrophy (HFMD) during preclinical cardiomyopathy using TDI. The study animals included 22 healthy controls and 7 cats belonging to a family of cats with HFMD (2 affected adult males, 2 heterozygous adult females, one 2.5-month-old affected male kitten, and 2 phenotypically normal female kittens from the same litter). All cats were examined via conventional echocardiography and 2-dimensional color TDI. No LVFW hypertrophy was detected in the 2 carriers or in the affected kitten when using conventional echocardiography and histologic examination, respectively. The LVFW also was normal for 1 affected male and at the upper limit of normal for the 2nd male. Conversely, LVFW dysfunction was detected in all affected and carrier cats with HFMD when using TDI. TDI consistently detects LVFW dysfunction in cats with HFMD despite the absence of myocardial hypertrophy. Therefore, TDI appears more sensitive than conventional echocardiography in detecting regional myocardial abnormalities.