Generation of mature compact ventricular cardiomyocytes from human pluripotent stem cells.

Compact cardiomyocytes that make up the ventricular wall of the adult heart represent an important therapeutic target population for modeling and treating cardiovascular diseases. Here, we established a differentiation strategy that promotes the specification, proliferation and maturation of compact ventricular cardiomyocytes from human pluripotent stem cells (hPSCs). The cardiomyocytes generated under these conditions display the ability to use fatty acids as an energy source, a high mitochondrial mass, well-defined sarcomere structures and enhanced contraction force. These ventricular cells undergo metabolic changes indicative of those associated with heart failure when challenged in vitro with pathological stimuli and were found to generate grafts consisting of more mature cells than those derived from immature cardiomyocytes following transplantation into infarcted rat hearts. hPSC-derived atrial cardiomyocytes also responded to the maturation cues identified in this study, indicating that the approach is broadly applicable to different subtypes of the heart. Collectively, these findings highlight the power of recapitulating key aspects of embryonic and postnatal development for generating therapeutically relevant cell types from hPSCs.

A new anti-Helicobacter pylori juglone from Reynoutria japonica.

A 70% ethanol extract from the root portion of Reynoutria japonica afforded one new and three known juglone derivatives, namely, 2-methoxy-6-acetyl-7-methyljuglone (1), 2-ethoxy-6-acetyl-7-methyljuglone (2), 2-methoxy-7-acetonyljuglone (3), and 3-acetyl-7-methoxy-2-methyljuglone (4) together with two phenolics (5 and 6), an anthraquinone (7), a stilbene (8) and a phthalide (9). Their structures were elucidated on the basis of comprehensive spectroscopic studies including IR, MS, and 1H, 13C, 2D NMR spectra. Compound 3 is a new compound in nature, and compounds 4-6 have been isolated for the first time from R. japonica. The isolates were evaluated for their antibacterial activity against three strains (43504, 51, and 26695) of Helicobacter pylori. The four isolated juglone derivatives (1-4) showed potent growth inhibitory activity. Among them, compounds 1-3 exhibited stronger inhibitory activity than those of the positive controls, juglone and metronidazole, for the three strains and that of another reference, clarithromycin, for the 43504 and 51 strains. Specifically, the new juglone compound 3 displayed the most potent antibacterial activity against all three strains, 43504, 51, and 26695, with MIC values of 0.06, 0.06 and 0.13 µM, respectively, and MIC50 values of 0.14, 0.11 and 0.15 µM, respectively.

Modeling Atrial Fibrillation using Human Embryonic Stem Cell-Derived Atrial Tissue.

Since current experimental models of Atrial Fibrillation (AF) have significant limitations, we used human embryonic stem cells (hESCs) to generate an atrial-specific tissue model of AF for pharmacologic testing. We generated atrial-like cardiomyocytes (CMs) from hESCs which preferentially expressed atrial-specific genes, and had shorter action potential (AP) durations compared to ventricular-like CMs. We then generated confluent atrial-like CM sheets and interrogated them using optical mapping techniques. Atrial-like CM sheets (~1 cm in diameter) showed uniform AP propagation, and rapid re-entrant rotor patterns, as seen in AF could be induced. Anti-arrhythmic drugs were tested on single atrial-like CMs and cell sheets. Flecainide profoundly slowed upstroke velocity without affecting AP duration, leading to reduced conduction velocities (CVs), curvatures and cycle lengths of rotors, consistent with increased rotor organization and expansion. By contrast, consistent with block of rapid delayed rectifier K+ currents (Ikr) and AP prolongation in isolated atrial-like CMs, dofetilide prolonged APs and reduced cycle lengths of rotors in cell sheets without affecting CV. In conclusion, using our hESC-derived atrial CM preparations, we demonstrate that flecainide and dofetilide modulate reentrant arrhythmogenic rotor activation patterns in a manner that helps explain their efficacy in treating and preventing AF.

Complete Genome Sequences of Porcine Deltacoronavirus Strains DH1/2016 and DH2/2016 Isolated in South Korea.

Two porcine deltacoronavirus (PDCoV) strains, named DH1/2016 and DH2/2016, were isolated from feces of piglets which had severe watery diarrhea symptoms. A comparison of the complete genome sequences suggested that the DH1/2016 and DH2/2016 strains are highly homologous to each other and to PDCoVs isolated in early 2014 from the United States.

New emergence pattern with variant porcine epidemic diarrhea viruses, South Korea, 2012-2015.

Since outbreaks of porcine epidemic diarrhea virus (PEDV) in the United States in 2013, explosive outbreaks of PED in South Korea have infected all age groups of pigs in 2014-2015year. This study analyzed a large collection of the Spike protein coding gene to infer the spatial-temporal diffusion history of PEDV. The studying results suggested that PEDVs in Korea belonged to different genogroups. While classical G1 was continuingly circulating between provinces of Korea, the pandemic G2a were recently introduced from China and USA. By the application of Bayesian phylogeographical analysis, this study demonstrated the spatial-temporal transmission of PEDVs within Korea. Of the recent emerged G2a viruses, J3142 strains showed potential recombination breakpoint (376-2,143nt) of S1 gene between KNU1303_Korea strain_G2a (KJ451046) and 45RWVCF0712_Thailand strain_G2b (KF724935). The pandemic G2a virus was partial neutralized by the antibodies invoked by the G1- based PED vaccine virus.

Increased humoral antibody response of foot-and-mouth disease virus vaccine in growing pigs pre-treated with poly-γ-glutamic acid.

This study was conducted to determine if humoral antibody response of foot-and-mouth disease (FMD) vaccine improved in 8-week-old growing pigs born to well-vaccinated sows pre-treated with 60 mg of poly-γ-glutamic acid (γ-PGA) three days before vaccination. Antibody against FMD virus serotype O was measured 0, 2, 4 and 6 weeks post-vaccination, using a PrioCHECK FMDV type O ELISA kit. The results showed that positive antibody reactions against FMDV serotype O antigen among a component of the vaccine significantly increased in response to pre-injection with γ-PGA.

Comparative analyses of humoral and cell-mediated immune responses upon vaccination with different commercially available single-dose porcine circovirus type 2 vaccines.

The objective of this study was to compare the induction of humoral and cell-mediated immune responses by four commercially available single-dose porcine circovirus type 2 (PCV-2) vaccines. A total of 50 3-week-old piglets were assigned to five groups (10 pigs per group). Four commercial PCV-2 vaccines were administered according to the manufacturer's instructions and the piglets were observed for 154 days post vaccination (dpv). Inactivated chimeric PCV-1-2 vaccines induced higher levels of PCV-2-specific neutralizing antibodies (NA) and interferon-γ-secreting cells (IFN-γ-SC) in pigs than did the other three commercial PCV-2 vaccines. The proportions of CD4(+) cells were significantly higher in animals vaccinated with inactivated chimeric PCV-1-2 and PCV-2 vaccines than in animals vaccinated with the two subunit vaccines. To our knowledge, this is the first comparison of humoral and cell-mediated immunity induced by four commercial single-dose PCV-2 vaccines under the same conditions. The results of this study demonstrated quantitative differences in the induction of humoral and cell-mediated immunity following vaccination.

Digoxigenin-labeled in situ hybridization for the detection of Streptococcus suis DNA in polyserositis and a comparison with biotinylated in situ hybridization.

The objective of this study was to develop digoxigenin-labeled in situ hybridization (ISH) for the detection of Streptococcus suis in naturally infected pigs with polyserositis and to compare it with biotinylated ISH. Digoxigenin-labeled hybridization signals for S. suis were observed in cells that had infiltrated the fibrous polyserositis and microcolonies in the blood vessels. Mock hybridization showed no hybridization signals for endogenous digoxigenin. Biotinylated hybridization signals for S. suis were observed in cells that had infiltrated the fibrous polyserositis. However, similar hybridization signals were also observed in the fibrous inflammatory area using mock hybridization for endogenous biotin. The present study demonstrated that digoxigenin-labeled ISH is a valuable diagnostic tool for specific detection of S. suis in polyserositic tissues without nonspecific reactions compared with biotinylated ISH.

Inhibition of porcine endogenous retrovirus in PK15 cell line by efficient multitargeting RNA interference.

To effectively suppress porcine endogenous retroviruses (PERV)s, RNAi technique was utilized. RNAi is the up-to-date skill for gene knockdown which simultaneously multitargets both gag and pol genes critical for replication of PERVs. Previously, two of the most effective siRNAs (gag2, pol2) were found to reduce the expression of PERVs. Concurrent treatment of these two siRNAs (gag2+pol2) showed knockdown efficiency of up to 88% compared to negative control. However, despite the high initial knockdown efficiency 48 h after transfection caused by siRNA, it may only be a transient effect of suppressing PERVs. The multitargeting vector was designed, containing both gag and pol genes and making use of POL II miR Expression Vector, which allowed for persistent and multiple targeting. This is the latest shRNA system technique expressing and targeting like miRNA. Through antibiotics resistance characteristics utilizing this vector, miRNA-transfected PK15 cells (gag2-pol2) were selected during 10 days. An 88.1% reduction in the level of mRNA expression was found. In addition, we performed RT-activity analysis and fluorescence in situ hybridization assay, and it demonstrated the highest knockdown efficiency in multitargeting (gag2+pol2) miRNA group. Therefore, according to the results above, gene knockdown system (siRNA and shRNA) through multitargeting strategy could effectively inhibit PERVs.

Optimized protocol for multiplex nested polymerase chain reaction to detect and differentiate Haemophilus parasuis, Streptococcus suis, and Mycoplasma hyorhinis in formalin-fixed, paraffin-embedded tissues from pigs with polyserositis.

An optimized protocol was developed for the simultaneous detection and differentiation of Haemophilus parasuis, Streptococcus suis, and Mycoplasma hyorhinis in formalin-fixed, paraffin-embedded (FFPE) tissues with multiplex nested polymerase chain reaction (PCR). This method also determines the prevalence of these bacteria in pigs with polyserositis. DNA extraction with a combination of a commercial reagent and proteinase K resulted in more frequent detection of the pathogens than DNA extraction with proteinase K alone. Among FFPE tissue samples from 312 cases of polyserositis in which at least 1 bacterial species was detected, multiplex nested PCR detected H. parasuis in 239 (77%), S. suis in 124 (40%), and M. hyorhinis in 40 (13%). The disease was caused by a single pathogen in 224 (72%) of the cases and multiple pathogens in 88 (28%). Among the pigs positive for H. parasuis, S. suis, and M. hyorhinis by multiplex nested PCR, the pathogen was isolated from only 11%, 35%, and 28%, respectively. Therefore, the PCR protocol developed in this study is a useful diagnostic method when samples are negative after isolation methods and even for samples in which only 1 pathogen was isolated.

Development of in situ hybridization for the detection of Mycoplasma hyorhinis in formalin-fixed paraffin-embedded tissues from naturally infected pigs with polyserositis.

The aim of this study was to develop in situ hybridization for detection of Mycoplasma hyorhinis in formalin-fixed, paraffin-wax-embedded tissues from pigs with polyserositis. M. hyorhinis was isolated from the spleen (2 pigs) and pericardium (1 pig). M. hyorhinis DNA was detected 16 out of 20 pigs with polyserositis. In situ hybridization produced a distinct positive signal for the M. hyorhinis p37 gene in inflammatory cells in the polyserositis. In situ hybridization developed in the present study present diagnostic tools capable of detection of M. hyorhinis in formalin-fixed, paraffin-wax-embedded tissues from the naturally infected pigs.

Distribution of capsular serotypes and virulence markers of Streptococcus suis isolated from pigs with polyserositis in Korea.

The objective of this study was to determine the capsular serotypes and potential virulence factors of Streptococcus suis isolated from pigs with polyserositis. Among the 24 isolates evaluated, serotype 3 [7 (29%) of the isolates] and serotype 4 [5 (21%)] were the most common. The isolates were also studied for the presence of the genes mrp, epf, and sly, which encode muramidase-released protein (MRP), extracellular factor (EF), and suilysin (SLY), respectively. Of the 24 isolates, 8 carried mrp: 4 of serotype 3, 2 of serotype 2, and 2 of serotype 4. One mrp(+) isolate (serotype 2) also carried the epf gene. All 24 isolates carried the sly gene. The serotype and genotype distribution greatly differed from that reported for isolates from pigs with other clinical manifestations of S. suis infection in other countries.

Noninvasive biosignal detection radar system using circular polarization.

This paper proposes an integrated hypersensitive Doppler radar system through a circular polarization characteristic. Through the idea of a reverse sense of rotation when the reflecting surface is perfectly conducting, it is shown that the detecting property of the system can be effectively improved by using antennas that have a reverse polarization. This bistatic radar system can be used in noninvasively sensing biosignals such as respiration and heart rates with the periodic movement of skin and muscle near the heart. The operating frequency of the system is in the X-band and the radar size is 95 x50 x13 mm(3).