Oligos for Real-Time RT-PCR experiments.

<p>Oligos for Real-Time RT-PCR experiments.</p

Up-regulation of PDCD5 in Ang II-induced cardiac hypertrophy.

<p>(A), HW∶BW ratio showing significant increase in cardiac mass in mice treated with Ang II for 2 weeks compared to sham control mice. (B) Expression of ANF was determined by quantitative Real-Time RT-PCR analysis in cDNA samples derived from hearts of mice treated with Ang II and sham control. (C), Expression of βMHC was determined by quantitative Real-Time RT-PCR analysis in cDNA samples derived from hearts of mice treated with Ang II and sham control. (D), Representative western blot of PDCD5 and internal control actin proteins in heart extracts from 8–week-old male mice with sham or Ang II treatment. (E), Quantitative analysis revealed that PDCD5 levels were up-regulated in hearts from mice treated with Ang II (n = 4) as compared to sham control mice (n = 4). *<i>P<0.05</i>, sham vs. Ang II treatment.</p

Assessment of autophagy in high over-expressing line.

<p>(A), Representative images of Immunostaining of LC3 on paraffin section of hearts from high over-expressing line and WT control mice. (B), Quantification of LC3 positive dots (For each group, 4 mice were studied). (C), Representative western blot of autophagy-related proteins LC3, Beclin 1, LAMP-1 and cathepsin D in heart extracts obtained from 3-month-old high over-expressing line and WT control mice. (D), Densitometric analysis of LC3 immunoblots. (E), Representative western blot of time course analysis of LC3 processing in heart extracts obtained from high over-expressing line and WT control mice. (F), Representative western blot of acetylated p53 and DRAM.</p

Echocardiographic analysis of cardiac function of high over-expressing line.

<p>(A), Representative M-mode echocardiography images in 3-month-old mice. (B)–(C), LV end-diastolic diameter (LVID;d) and LV end-systolic diameter (LVID;s) were significantly increased in high over-expressing line compared to WT control mice. (D), Left ventricular posterior wall (LVPW) was significantly decreased in high over-expressing line compared to WT control mice. (E)–(F), % fractional shortening and ejection fraction were significantly diminished in high over-expressing line compared to WT control mice. *<i>P</i><0.05, WT vs. TG (n = 5 for TG, n = 5 for WT, 3 months old).</p

Generation of transgenic mice with cardiac specific over-expression of hPDCD5.

<p>(A), Schematic representation of the transgene construct of hPDCD5 cDNA under the control of the cardiac-specific alpha-MHC promoter (not at scale). (B), Representative western blot of hPDCD5 protein with PDCD5 antibody in heart extracts from high over-expressing line and WT control mice. Asterisk indicates endogenous PDCD5. (C), Densitometric analysis of PDCD5 immunoblots. (D), Representative western blot of hPDCD5 protein with HA antibody in heart extracts obtained from high over-expressing line and WT control mice. (E), Representative western blot of hPDCD5 protein with HA antibody in heart, liver, and brain extracts obtained from high over-expressing line and WT control mice.</p

Cardiac remodeling in high PDCD5 over-expressing line.

<p>(A), Cardiac specific high over-expression of hPDCD5 results in enlarged hearts (3 months old). (B), HW∶BW ratio showing significant increase in cardiac mass in high over-expressing line compared to the WT control mice (n = 6 for TG, n = 6 for WT). (C), Measurement of two-dimensional cardiomyocyte cross-sectional area showing significantly enlarged cells in high over-expressing line compared with WT control mice (n = 4 for TG, n = 4 for WT). *<i>P</i><0.05, WT vs. TG. (D), Histological analysis with H&E staining on heart sections. Bar, 50 µm. (E), Masson's trichrome staining showing collagen depositon in left ventricle of high over-expressing line. Bar, 50 µm. Expression of ANF (F), BNP (G), SAA (H), αMHC (I), βMHC (J) and SERCA (K) was determined by quantitative Real-Time RT-PCR analysis in cDNA samples derived from heart of high over-expressing line and WT control mice (n = 4 for TG, n = 4 for WT). Expression levels were normalized to GAPDH. Experiments were performed twice in triplicate with similar results. *<i>P</i><0.05, WT vs. TG.</p

Susceptibility of low over-expressing line to Ang II-induced cardiac hypertrophy.

<p>(A), H&E staining on heart sections. Bar, 40 µm. (B), Measurement of two-dimensional cardiomyocyte cross-sectional area. (C), LV to BW ratio is significantly increased in Ang II- treated transgenic mice as compared to WT control mice receiving identical treatment. (D), In transgenic mice treated with Ang II, FS% is not significantly altered as compared to WT control mice. (E), Representative images of LC3 processing in Ang II-treated transgenic mice and WT control mice. (F) Densitometric analysis of LC3 immunoblots. *<i>P</i><0.05, WT vs. TG (n = 4–5 for each group).</p

The effect of hPDCD5 over-expression on the survival of mice.

<p>(A), Autopsy of transgenic founder 41 showing dramatically enlarged heart. (B), Kaplan-Meier Survival Curves of high over-expressing line (red line) and WT littermate controls (black line). (n = 50 for WT, n = 65 for TG).</p

Co-Circulation of the Rare CPV-2c with Unique Gln370Arg Substitution, New CPV-2b with Unique Thr440Ala Substitution, and New CPV-2a with High Prevalence and Variation in Heilongjiang Province, Northeast China

<div><p>To trace evolution of canine parvovirus-2 (CPV-2), a total of 201 stool samples were collected from dogs with diarrhea in Heilongjiang province of northeast China from May 2014 to April 2015. The presence of CPV-2 in the samples was determined by PCR amplification of the VP2 gene (568 bp) of CPV-2. The results revealed that 95 samples (47.26%) were positive for CPV-2, and they showed 98.8%–100% nucleotide identity and 97.6%–100% amino acid identity. Of 95 CPV-2-positive samples, types new2a (Ser297Ala), new2b (Ser297Ala), and 2c accounted for 64.21%, 21.05%, and 14.74%, respectively. The positive rate of CPV-2 and the distribution of the new2a, new2b and 2c types exhibited differences among regions, seasons, and ages. Immunized dogs accounted for 48.42% of 95 CPV-2-positive samples. Coinfections with canine coronavirus, canine kobuvirus, and canine bocavirus were identified. Phylogenetic analysis revealed that the identified new2a, new2b, and CPV-2c strains in our study exhibited a close relationship with most of the CPV-2 strains from China; type new2a strains exhibited high variability, forming three subgroups; type new2b and CPV-2c strains formed one group with reference strains from China. Of 95 CPV-2 strains, Tyr324Ile and Thr440Ala substitutions accounted for 100% and 64.21%, respectively; all type new2b strains exhibited the Thr440Ala substitution, while the unique Gln370Arg substitution was found in all type 2c strains. Recombination analysis using entire VP2 gene indicated possible recombination events between the identified CPV-2 strains and reference strains from China. Our data revealed the co-circulation of new CPV-2a, new CPV-2b, and rare CPV-2c, as well as potential recombination events among Chinese CPV-2 strains.</p></div

Phylogenetic analysis of CCoV strains on the basis of partial S gene sequences.

<p>(A) Phylogenetic tree generated using the nucleotide sequences of the partial S gene of CCoV-IIa. (B) Phylogenetic tree generated using the nucleotide sequences of the partial S gene of CCoV-I. (C) Phylogenetic tree generated using the nucleotide sequences of the partial S gene of CCoV-IIb. Red circles represent the CCoV strains identified in our study.</p