Selection of reference genes in canine uterine tissues.

Real-time quantitative polymerase chain reaction (RT-qPCR) is usually employed in gene expression studies in veterinary research, including in studies on canine pyometra. Canine pyometra is a common clinical disease in bitches. When using RT-qPCR, internal standards, such as reference genes, are necessary to investigate relative gene expression by quantitative measurements of mRNA levels. The aim of this study was to evaluate the stability of reference genes and select reference genes suitable for canine pyometra studies. We collected 24 bitch uterine tissue samples, including five healthy and 19 pyometra infected samples. These were used to screen the best reference genes of seven candidate genes (18SrRNA, ACTB, B2M, GAPDH, HPRT, RPL13A, and YWHAZ). The method of KH Sadek and the GeNorm, Normfinder, BestKeeper, and RefFinder software were used to evaluate the stability of gene expression in both pyometra and healthy uterine samples. The results showed that the expression stability of the candidate gene in pyometra and healthy tissues differed. We showed that YWHAZ was the best reference gene, which could be used as an accurate internal control gene in canine pyometra studies. To further validate this recommendation, the expression profile of a target gene insulin-like growth factor 1 receptor gene (IGF1R) was investigated. We found that the expression of IGF1R was significantly altered when different reference genes were used. All reference genes identified in the present study will enable more accurate normalization of gene expression data in both pyometra infected and healthy uterine tissues.

Molecular analysis of MLH1 variants in Chinese sporadic colorectal cancer patients.

Single nucleotide polymorphisms (SNPs) in mismatch repair genes, especially in the MLH1 gene, are closely associated with susceptibility to hereditary nonpolyposis colorectal cancer. However, few relevant findings are available regarding the association between sporadic colorectal cancer (SCRC) and SNPs of MLH1 in Chinese patients. Therefore, the present study aimed to describe the pathogenic association between three important MLH1 polymorphisms and SCRC in the Chinese population. Peripheral blood samples from 156 SCRC patients and 311 healthy controls were collected. DNA was purified from peripheral blood, and the V384D, R217C, and I219V polymorphisms were evaluated using high-resolution melting analysis and direct sequencing. The association between the three important MLH1 polymorphisms and clinical pathological features of the SCRC patients was analyzed. In addition, PMS2-MLH1 protein interactions were determined by co-immunoprecipitation (Co-IP) to determine the protein functional alteration induced by these SNPs. Among the three polymorphisms, V384D was significantly associated with the risk of SCRC (OR = 31.36, P < 0.0001). The allele frequencies were 4.81 and 0.16% in the SCRC group. No association was found between SCRC and R217C, or between SCRC and I219V. Moreover, the allele frequency of R217C was significantly higher in the SCRC patients younger than 60 years than in those older than 60 years. Co-IP showed that the MLH1 R217C, V384D, and I219V variants had relative binding abilities with PMS2 of 0.59, 0.70, and 0.80, respectively, compared with the wild-type. These findings suggest that MLH1 V384D could be a promising genetic marker for susceptibility to SCRC.

Disentangling the roles of chance, abiotic factors and biotic interactions among epiphytic bryophyte communities in a tropical rainforest (Yunnan, China).

Epiphytes offer an appealing framework to disentangle the contributions of chance, biotic and abiotic drivers of species distributions. In the context of the stress-gradient theory, we test the hypotheses that (i) deterministic (i.e., non-random) factors play an increasing role in communities from young to old trees, (ii) negative biotic interactions increase on older trees and towards the tree base, and (iii) positive interactions show the reverse pattern. Bryophyte species distributions and abiotic conditions were recorded on a 1.1 ha tropical rainforest canopy crane site. We analysed co-occurrence patterns in a niche modelling framework to disentangle the roles of chance, abiotic factors and putative biotic interactions among species pairs. 76% of species pairs resulted from chance. Abiotic factors explained 78% of non-randomly associated species pairs, and co-occurrences prevailed over non-coincidences in the remaining species pairs. Positive and negative interactions mostly involved species pairs from the same versus different communities (mosses versus liverworts) and life forms, respectively. There was an increase in randomly associated pairs from large to small trees. No increase in negative interactions from young to old trees or from the canopy to the base was observed. Our results suggest that epiphytic bryophyte community composition is primarily driven by environmental filtering, whose importance increases with niche complexity and diversity. Biotic interactions play a secondary role, with a very marginal contribution of competitive exclusion. Biotic interactions vary among communities (mosses versus liverworts) and life forms, facilitation prevailing among species from the same community and life form, and competition among species from different communities and life forms.

Prevalence of arrhythmias in patients with idiopathic dilated cardiomyopathy.

A total of 105 patients with idiopathic dilated cardiomyopathy (IDCM) underwent Holter monitoring. The prevalence of arrhythmias was as follows: ventricular premature beats (VPB) 100%, including complex VPB 58.1%, short runs of ventricular tachycardia (VT) 25.7%, A-VB or BBB 46.7%, atrial arrhythmias 38.1%, sinus node dysfunction 6.6%, ST-T change 38.1%. The incidence of ventricular arrhythmia (VA) was not related to severity of cardiac dysfunction (NYHA), duration of illness and sex. The most common arrhythmia was VA, the second one was heart block and atrial arrhythmia. Serum norepinephrine and epinephrine levels were 743.4 +/- 252.5 and 688.0 +/- 452.4 pg/ml, respectively, which were higher than those in control group (P < 0.01). Isoproterenol sensitivity test (ICD25) 8.43 +/- 11.21 micrograms, was also much higher than that in control group (P < 0.01). Average H-V interval was 69.4 +/- 13.3 msec in HBE. Eight patients died. Two died of congestive heart failure (class II-IV), and six cases were diagnosed as sudden cardiac death resulting from VT and VF.

Experimental study on the occlusion of arterial blood flow by the implantation of nitinol alloy stents.

To evaluate the occlusion of arteries, 6 conic intravascular endoprosthetic stents of titanium-nickel alloy were implanted transluminally to the right external iliac arteries in 2 normal dogs and 4 normal pigs. The stent was characterized by ductility below 313 K (40 degrees C) and restoration memory-shape between 313-319 K. The diameter of the large end of conic stent was 4.0 mm (dog) and 4.5 mm(pig). The topographic anatomy showed that the external iliac artery was near complete occlusion, with a very small residual lumen (about 1 mm in diameter) in dogs at 6 months, with complete occlusion in pigs at 8 months. The histopathological examination indicated that the arterial intima of the upper branch from the stented site was smooth, and that neither inflammatory cell infiltration nor foreign giant cell reaction was found. The newly formed granulation tissue inside the stent came from emboli organization. These results suggest that the shape-memory alloy stent has good biocompatibility and the conic intravascular endoprosthetic stent might completely occlude arterial blood flow.

[The changes in beta-adrenergic receptors in the course of dilated cardiomyopathy].

The relation between the density of beta receptors on myocardial cells and lymphocytes and the pathologic change of myocardium in hypertensive-diabetic (HD) rats in various stages during development of dilated cardiomyopathy (DCM) was investigated and the alteration of lymphocytic beta receptors in 20 patients with DCM was also studied. The results showed that the density of beta receptors on lymphocytes in the patients with class I-II cardiac status appeared to be in up regulation. The same was found with the receptors on lymphocytes and myocardial cells in HD rats at early stage. Conversely, the receptors were in down regulation on lymphocytes in DCM patients with class III-IV cardiac status and the same with those on lymphocytes and myocardial cells in HD rats at late stage. The changes of the receptors on lymphocytes and myocardial cells in the rats and the patients were quite parallel. Up regulation of beta receptors happened earlier than the pathologic changes in the myocardium in HD rats. Thus, the changes of lymphocytic beta receptors did reflect the behaviour of the receptors on myocardial cells with these data, it is reasonable to use beta blocker at early stage in patients with DCM so as to slow down the disease process.

[Changes in the relaxation and compliance of the left ventricle in cardiomyopathy].

In order to study the left ventricular diastolic events in cardiomyopathy, carotid pulse tracing, electrocardiogram, phonocardiogram and apexcardiogram with its first derivatives (dA/dt) were simultaneously recorded in 240 normal subjects, 30 patients with dilated cardiomyopathy (DCM) and 44 patients with hypertrophic cardiomyopathy (HCM). Total relaxation time (TRT), total relaxation time index (TRTI), the ratio of A wave to total diastolic amplitude in apexcardiogram (A/D), diastolic amplitude time index (DATI), the ratio of RF wave in apexcardiogram to the total amplitude of apex tracing (F/H) and the variables reflecting the diastolic properties were measured to evaluate the left ventricular relaxation and compliance. Meanwhile, two variables reflecting the contractile ability of the left ventricle were measured in order to compare with the diastolic events. The results demonstrated that in normal subjects relaxation and compliance of the left ventricle showed a tendency of natural "decay" with aging and in patients with DCM and HCM these were significantly decreased, especially in patients with HCM. A/D, which reflects the left ventricular compliance, was increased in patients with HCM as compared with normal subjects (P less than 0.01) and also with patients with DCM. It means that a greater A/D is in favor of the diagnosis of HCM rather than DCM. In addition to abnormal diastolic events, systolic dysfunction, as evidenced by remarkable abnormalities of the contractile variables (PEP/LVET and R-dA/dt.I), was found as well in patients with DCM.

[Studies on the genetic susceptibility to dilated cardiomyopathy].

HLA-DQB1,-DRB1 genes of 27 Chinese patients with dilated cardiomyopathy (DCM), 7 high risk individuals in a DCM kindred and 17 normal control subjects were analysed with the use of restriction fragment length polymorphisms (RFLP) with full length DQB1 and DRB1 cDNA probes according to the standard and nomenclature of the Xth International Histocompatibility Workshop. The resulting restriction patterns allowed genotyping of HLA-DR and HLA-DQw. D-DQw8 frequency increased significantly in patients with DCM as compared with that of the controls (P < 0.05). D-DQw4 also increased in patients although no statistical significance was shown when Chi-square value was corrected with Yate's correction, whereas D-DQw5 overrepresented in controls (P < 0.05). Over half of the high risk individuals (4/7) in the familial DCM kindred carry D-DQw8 and D-DQw4. These results support the hypothesis that HLA class II genes were associated with an increased risk for DCM, HLA-DQB rather than -DRB may confer genetic susceptibility to DCM.

Experimental study of bone marrow-derived mesenchymal stem cells combined with hepatocyte growth factor transplantation via noninfarct-relative artery in acute myocardial infarction.

We investigated the impact of bone marrow-derived mesenchymal stem cells (BM-MSCs) alone or in combination with hepatocyte growth factor (HGF) transplantation via noninfarct-relative artery in a swine myocardial infarction (MI) model. Donor BM-MSCs were derived in vitro from swine auto-bone marrow cultures labeled by bromodeoxyuridine (BrdU) incorporation. Host MI swine model was created by ligating the distal left anterior descending artery. After 4 weeks, age-matched male MI swines were used for the transplantation. Male MI swines were transfused via noninfarct-relative artery with vehicle (control, n=6) or BrdU-labeled BM-MSCs (5 x 10(6)) alone (MSCs, n=6) or BrdU-labeled BM-MSCs (5 x 10(6)) combined with HGF (4 x 10(9) PFU) (MSCs+HGF, n=6). To evaluate the collateral artery growth (Rentrop) and cardiac perfusion in these animals, gate cardiac perfusion imaging and coronary angiography were performed before and 4 weeks after transplantation, respectively. To assess the contribution of donor-originated cells in stimulation of cardiomyocyte regeneration and angiogenesis, immunohistochemistry for BrdU and alpha-smooth muscle actin (alpha-SMA) and quantitative image analysis were performed at 4 weeks after transplantation. The results are as follows: (1) BrdU-positive cells were detected in host myocardium in both MSCs and MSCs+HGF groups, but not in the vehicle group. Most BrdU-positive cells expressed myosin heavy chain beta. (2) alpha-SMA(-)positive arteriole densities in the infarcted border area and infarcted area were increased significantly in both transplantation groups compared with the vehicle group. (3) Gate cardiac perfusion imaging demonstrated that the cardiac perfusion was significantly improved in transplantation groups compared with the vehicle group. (4) Ejection fraction and alpha-SMA-positive arteriole densities were increased significantly in both transplantation groups compared with the vehicle group. However, there was no difference in ejection fraction and alpha-SMA-positive arteriole densities between the MSCs group and the MSCs+HGF group. Growth of collateral arteries was not detected by coronary angiography in all three groups. In conclusion, the current study indicates that BM-MSCs transplantation via noninfarct-relative artery stimulates cardiomyocyte regeneration and angiogenesis and improves cardiac function, but does not stimulate collateral artery growth. BM-MSCs transplantation combined with HGF therapy is not superior to BM-MSCs alone transplantation. BM-MSCs transplantation via noninfarct-relative artery may be an alternative for those patients who cannot be transplanted via infarct-relative artery in clinical practice.