Rationale

Abnormal behavior of the cardiac ryanodine receptor (RyR2) has been linked to cardiac arrhythmias and heart failure (HF) after Myocardial infarction (MI). It has been proposed that protein kinase A (PKA) hyperphosphorylation of the RyR2 at a single residue, Ser-2808 is a critical mediator of RyR dysfunction, depressed cardiac performance and HF after MI.

Objectives

We used a mouse model (RyRS2808A) in which PKA hyperphosphorylation of the RyR2 at Ser-2808 is prevented to determine whether loss of PKA phosphorylation at this site averts post MI cardiac pump dysfunction.

Methods and Results

MI was induced in WT and S2808A mice. Myocyte and cardiac function were compared in WT and S2808A animals before and after MI. The effects of the PKA activator Isoproterenol (Iso) on L-type Ca2+ current (ICaL), contractions and [Ca2+]I transients were also measured. Both WT and S2808A mice had depressed pump function after MI and were no differences between groups. MI size was also identical in both groups. L type Ca2+ current, contractions, Ca2+ transients and SR Ca2+ load were also not significantly different in WT versus S2808A myocytes either before or after MI. Iso effects on Ca2+ current, contraction, Ca2+ transients and SR Ca2+ load were identical in WT and S2808A myocytes before and after MI at both low and high concentrations.

Conclusions

These results strongly support the idea that PKA phosphorylation of RyR-S2808 is irrelevant to the development of cardiac dysfunction after MI, at least in the mice used in this study.

Rationale

The source of Ca2+ to activate pathological cardiac hypertrophy is not clearly defined. Ca2+ influx through the L-type Ca2+ channels (LTCCs) determines “contractile” Ca2+, which is not thought to be the source of “hypertrophic” Ca2+. However, some LTCCs are housed in caveolin-3 (Cav-3) enriched signaling microdomains and are not directly involved in contraction. The function of these LTCCs is unknown.

Objective

To test the idea that LTCCs in Cav-3 containing signaling domains are a source of Ca2+ to activate the calcineurin-nuclear factor of activated T cells (Cn-NFAT) signaling cascade that promotes pathological hypertrophy.

Methods and Results

We developed reagents that targeted Ca2+ channel blocking Rem proteins to Cav-3 containing membranes, which house a small fraction of cardiac LTCCs. Blocking LTCCs within this Cav-3 membrane domain eliminated a small fraction of the LTCC current, almost all of the Ca2+ influx induced NFAT nuclear translocation, but did not reduce myocyte contractility.

Conclusions

We provide proof of concept that Ca2+ influx through LTCCs within caveolae signaling domains can activate “hypertrophic” signaling, and this Ca2+ influx can be selectively blocked without reducing cardiac contractility.

Gut microbiota has diverse ecological and evolutionary effects on its hosts. However, the ways in which it responds to environmental heterogeneity and host physiology remain poorly understood. To this end, we surveyed intestinal microbiota of Holotrichia parallela larvae at different instars and from different geographic regions. Bacterial 16S rRNA gene clone libraries were constructed and clones were subsequently screened by DGGE and sequenced. Firmicutes and Proteobacteria were the major phyla, and bacteria belonging to Ruminococcaceae, Lachnospiraceae, Enterobacteriaceae, Desulfovibrionaceae and Rhodocyclaceae families were commonly found in all natural populations. However, bacterial diversity (Chao1 and Shannon indices) and community structure varied across host populations, and the observed variation can be explained by soil pH, organic carbon and total nitrogen, and the climate factors (e.g., mean annual temperature) of the locations where the populations were sampled. Furthermore, increases in the species richness and diversity of gut microbiota were observed during larval growth. Bacteroidetes comprised the dominant group in the first instar; however, Firmicutes composed the majority of the hindgut microbiota during the second and third instars. Our results suggest that the gut's bacterial community changes in response to environmental heterogeneity and host's physiology, possibly to meet the host's ecological needs or physiological demands.

The Wnt signaling pathway plays an important role not only in embryonic development but also in the maintenance and differentiation of the stem cells in adulthood. In particular, Wnt signaling has been shown as an important regulatory pathway in the osteogenic differentiation of mesenchymal stem cells. Induction of the Wnt signaling pathway promotes bone formation while inactivation of the pathway leads to osteopenic states. Our current understanding of Wnt signaling in osteogenesis elucidates the molecular mechanisms of classic osteogenic pathologies. Activating and inactivating aberrations of the canonical Wnt signaling pathway in osteogenesis results in sclerosteosis and osteoporosis respectively. Recent studies have sought to target the Wnt signaling pathway to treat osteogenic disorders. Potential therapeutic approaches attempt to stimulate the Wnt signaling pathway by upregulating the intracellular mediators of the Wnt signaling cascade and inhibiting the endogenous antagonists of the pathway. Antibodies against endogenous antagonists, such as sclerostin and dickkopf-1, have demonstrated promising results in promoting bone formation and fracture healing. Lithium, an inhibitor of glycogen synthase kinase 3β, has also been reported to stimulate osteogenesis by stabilizing β catenin. Although manipulating the Wnt signaling pathway has abundant therapeutic potential, it requires cautious approach due to risks of tumorigenesis. The present review discusses the role of the Wnt signaling pathway in osteogenesis and examines its targeted therapeutic potential.

A series of angucyclinone antibiotics have been isolated from marine Streptomyces sp. strain W007 and identified. Here, a draft genome sequence of Streptomyces sp. W007 is presented. The genome contains an intact biosynthetic gene cluster for angucyclinone antibiotics, which provides insight into the combinatorial biosynthesis of angucyclinone antibiotics produced by marine streptomycetes.

Background

Locoregional recurrence is the typical pattern of recurrence in gastric cancer, and cannot be removed by surgery in most of the patients. We aimed to evaluate the feasibility and efficacy of computed tomography (CT)-guided brachytherapy for patients with locoregional recurrent gastric cancer.

Materials and methods

We reviewed the case histories of 28 patients with locoregional recurrent gastric cancer that were selected for CT- guided brachytherapy by a multidisciplinary team. The clinical data of the patients including patient characteristics, treatment parameters, short-term effects, and survival data were collected and analyzed.

Results

15-75 125I seeds were implanted into each patient to produce a minimal peripheral dose (MPD) 100-160 Gy. Median day 0 dosimetry was significant for the following: V100 (the volume treated with the prescription dose) 95.8% (90.2-120.5%) and D90 (prescription dose received by at least 90% of the volume) 105.2% (98.0-124.6%) of prescription dose. No serious complications occurred during the study. Two months after brachytherapy, complete response, partial response and progressive disease were observed in 50.0%, 28.6% and 21.4% of patients, respectively. The median survival time was 22.0 ± 5.2 months, and the 1, 2,and 3-year survival rate was 89 ± 6%, 52 ± 10% and 11 ± 7%, respectively. A univariate analysis showed that the tumor size was a significant predictor of overall survival (P = 0.034). Patients with tumors <3 cm had relatively higher complete response rate (66.7%), compared to those with tumors >3 cm (30.8%). The PTV (planning target volume) smaller than 45 cm3 was significantly correlated with achieving complete tumor eradication in the treated region (P = 0.020).

Conclusions

For selected patients with limited locoregional recurrent gastric cancer, CT-guided brachytherapy using 125I seeds implantation can provide a high local control rate, with minimal trauma.

The T-type Ca2+ channel (TTCC) plays important roles in cellular excitability and Ca2+ regulation. In the heart, TTCC is found in the sinoatrial nodal (SAN) and conduction cells. Cav3.1 encodes one of the three types of TTCCs. To date, there is no report regarding the regulation of Cav3.1 by β-adrenergic agonists, which is the topic of this study. Ventricular myocytes (VMs) from Cav3.1 double transgenic (TG) mice and SAN cells from wild type, Cav3.1 knockout, or Cav3.2 knockout mice were used to study β-adrenergic regulation of overexpressed or native Cav3.1-mediated T-type Ca2+ current (ICa-T(3.1)). ICa-T(3.1) was not found in control VMs but was robust in all examined TG-VMs. A β-adrenergic agonist (isoproterenol, ISO) and a cyclic AMP analog (dibutyryl-cAMP) significantly increased ICa-T(3.1) as well as ICa-L in TG-VMs at both physiological and room temperatures. The ISO effect on ICa-L and ICa-T in TG myocytes was blocked by H89, a PKA inhibitor. ICa-T was detected in control wildtype SAN cells but not in Cav3.1 knockout SAN cells, indicating the identity of ICa-T in normal SAN cells is mediated by Cav3.1. Real-time PCR confirmed the presence of Cav3.1 mRNA but not mRNAs of Cav3.2 and Cav3.3 in the SAN. ICa-T in SAN cells from wild type or Cav3.2 knockout mice was significantly increased by ISO, suggesting native Cav3.1 channels can be upregulated by the β-adrenergic (β-AR) system. In conclusion, β-adrenergic stimulation increases ICa-T(3.1) in cardiomyocytes, which is mediated by the cAMP/PKA pathway. The upregulation of ICa-T(3.1) by the β-adrenergic system could play important roles in cellular functions involving Cav3.1.

Background

DNA methylation plays important biological roles in plants and animals. To examine the rice genomic methylation landscape and assess its functional significance, we generated single-base resolution DNA methylome maps for Asian cultivated rice Oryza sativa ssp. japonica, indica and their wild relatives, Oryza rufipogon and Oryza nivara.

Results

The overall methylation level of rice genomes is four times higher than that of Arabidopsis. Consistent with the results reported for Arabidopsis, methylation in promoters represses gene expression while gene-body methylation generally appears to be positively associated with gene expression. Interestingly, we discovered that methylation in gene transcriptional termination regions (TTRs) can significantly repress gene expression, and the effect is even stronger than that of promoter methylation. Through integrated analysis of genomic, DNA methylomic and transcriptomic differences between cultivated and wild rice, we found that primary DNA sequence divergence is the major determinant of methylational differences at the whole genome level, but DNA methylational difference alone can only account for limited gene expression variation between the cultivated and wild rice. Furthermore, we identified a number of genes with significant difference in methylation level between the wild and cultivated rice.

Conclusions

The single-base resolution methylomes of rice obtained in this study have not only broadened our understanding of the mechanism and function of DNA methylation in plant genomes, but also provided valuable data for future studies of rice epigenetics and the epigenetic differentiation between wild and cultivated rice.

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER), nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.

Rationale

The ability of the adult heart to generate new myocytes after injury is not established.

Objective

Our purpose was to determine if the adult heart has the capacity to generate new myocytes after injury, and to gain insight into their source.

Methods and Results

Cardiac injury was induced in the adult feline heart by infusing Isoproterenol (ISO) for 10 days via minipumps and then animals were allowed to recover for 7 or 28 days. Cardiac function was measured with echocardiography and proliferative cells were identified by nuclear incorporation of 5-bromodeoxyuridine (BrdU; 7 day minipump infusion). BrdU was infused for 7 days before euthanasia at Days 10, 17, and 38 or during injury and animals euthanized at Day 38. ISO caused reduction in cardiac function with evidence of myocyte loss from necrosis. During this injury phase there was a significant increase in the number of proliferative cells in the atria and ventricle, but there was no increase in BrdU+ myocytes. cKit+ cardiac progenitor cells were BrdU labeled during injury. During the first seven days of recovery there was a significant reduction in cellular proliferation (BrdU incorporation) but a significant increase in BrdU+ myocytes. There was modest improvement in cardiac structure and function during recovery. At day 38, overall cell proliferation was not different than control but increased numbers of BrdU+ myocytes were found when BrdU was infused during injury.

Conclusions

These studies suggest that ISO injury activates cardiac progenitor cells that can differentiate into new myocytes during cardiac repair.

Background

Tephritid fruit flies in the genus Bactrocera are of major economic significance in agriculture causing considerable loss to the fruit and vegetable industry. Currently, there is no ideal control program. Molecular means is an effective method for pest control at present, but genomic or transcriptomic data for members of this genus remains limited. To facilitate molecular research into reproduction and development mechanisms, and finally effective control on these pests, an extensive transcriptome for the oriental fruit fly Bactrocera dorsalis was produced using the Roche 454-FLX platform.

Results

We obtained over 350 million bases of cDNA derived from the whole body of B. dorsalis at different developmental stages. In a single run, 747,206 sequencing reads with a mean read length of 382 bp were obtained. These reads were assembled into 28,782 contigs and 169,966 singletons. The mean contig size was 750 bp and many nearly full-length transcripts were assembled. Additionally, we identified a great number of genes that are involved in reproduction and development as well as genes that represent nearly all major conserved metazoan signal transduction pathways, such as insulin signal transduction. Furthermore, transcriptome changes during development were analyzed. A total of 2,977 differentially expressed genes (DEGs) were detected between larvae and pupae libraries, while there were 1,621 DEGs between adults and larvae, and 2,002 between adults and pupae. These DEGs were functionally annotated with KEGG pathway annotation and 9 genes were validated by qRT-PCR.

Conclusion

Our data represent the extensive sequence resources available for B. dorsalis and provide for the first time access to the genetic architecture of reproduction and development as well as major signal transduction pathways in the Tephritid fruit fly pests, allowing us to elucidate the molecular mechanisms underlying courtship, ovipositing, development and detailed analyses of the signal transduction pathways.

Background

Crosstalk between peripheral nociceptors belonging to the transient receptor potential vanilloid receptor subtype-1 (TRPV1) and ankyrin subtype-1 (TRPA1) family has recently been demonstrated. Moreover, the intravenous anesthetic propofol has been shown to directly activate TRPA1 receptors, and indirectly restore sensitivity of TRPV1 receptors in dorsal root ganglion (DRG) sensory neurons. Our objective was to determine the extent to which TRPA1 activation is involved in mediating the propofol-induced restoration of TRPV1 sensitivity.

Methods

Mouse DRG neurons were isolated by enzymatic dissociation and grown for 24 h. F-11 cells were transfected with complementary DNA for both TRPV1 and TRPA1 or TRPV1 only. Intracellular Ca2+ concentration was measured in individual cells via fluorescence microscopy. Following TRPV1 de-sensitization with capsaicin (100 nM), cells were treated with propofol (1, 5 and 10 μM) alone, propofol in the presence of the TRPA1 antagonist, HC-030031 (0.5 μM) or the TRPA1 agonist, Allyl isothiocyanate (AITC, 100 μM) and capsaicin was then reapplied.

Results

In DRG neurons that contain both TRPV1 and TRPA1, propofol and AITC restored TRPV1 sensitivity. However, in DRG neurons containing only TRPV1 receptors, exposure to propofol or AITC following de-sensitization did not restore capsaicin-induced TRPV1 sensitivity. Similarly, in F-11 cells transfected with both TRPV1 and TRPA1, propofol and AITC restored TRPV1 sensitivity. However, in F-11 cells transfected with TRPV1 only, neither propofol nor AITC were capable of restoring TRPV1 sensitivity.

Conclusions

These data demonstrate that propofol restores TRPV1 sensitivity in primary DRG neurons and in cultured F-11 cells transfected with both the TRPV1 and TRPA1 receptors via a TRPA1-dependent process. Propofol’s effects on sensory neurons may be clinically important and contribute to peripheral sensitization to nociceptive stimuli in traumatized tissue.

Over-activation of AMPARs (α−amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype glutamate receptors) is implicated in excitotoxic neuronal death associated with acute brain insults, such as ischemic stroke. However, the specific molecular mechanism by which AMPARs, especially the calcium-impermeable AMPARs, induce neuronal death remains poorly understood. Here we report the identification of a previously unrecognized molecular pathway involving a direct protein-protein interaction that underlies GluR2-containing AMPAR-mediated excitotoxicity. Agonist stimulation of AMPARs promotes GluR2/GAPDH (glyceraldehyde-3-phosphate dehydrogenase) complex formation and subsequent internalization. Disruption of GluR2/GAPDH interaction by administration of an interfering peptide prevents AMPAR-mediated excitotoxicity and protects against damage induced by oxygen-glucose deprivation (OGD), an in vitro model of brain ischemia.

Purpose

There is a need to supplement or supplant the conventional diagnostic tools, namely, cystoscopy and B-type ultrasound, for bladder cancer (BC). We aimed to identify novel DNA methylation markers for BC through genome-wide profiling of BC cell lines and subsequent methylation-specific PCR (MSP) screening of clinical urine samples.

Experimental Design

The methyl-DNA binding domain (MBD) capture technique, methylCap/seq, was performed to screen for specific hypermethylated CpG islands in two BC cell lines (5637 and T24). The top one hundred hypermethylated targets were sequentially screened by MSP in urine samples to gradually narrow the target number and optimize the composition of the diagnostic panel. The diagnostic performance of the obtained panel was evaluated in different clinical scenarios.

Results

A total of 1,627 hypermethylated promoter targets in the BC cell lines was identified by Illumina sequencing. The top 104 hypermethylated targets were reduced to eight genes (VAX1, KCNV1, ECEL1, TMEM26, TAL1, PROX1, SLC6A20, and LMX1A) after the urine DNA screening in a small sample size of 8 normal control and 18 BC subjects. Validation in an independent sample of 212 BC patients enabled the optimization of five methylation targets, including VAX1, KCNV1, TAL1, PPOX1, and CFTR, which was obtained in our previous study, for BC diagnosis with a sensitivity and specificity of 88.68% and 87.25%, respectively. In addition, the methylation of VAX1 and LMX1A was found to be associated with BC recurrence.

Conclusions

We identified a promising diagnostic marker panel for early non-invasive detection and subsequent BC surveillance.

Pathological cardiac hypertrophy (PCH) is associated with the development of arrhythmia and congestive heart failure. While calcium (Ca2+) is implicated in hypertrophic signaling pathways, the specific role of Ca2+ influx through the L-type Ca2+ channel (ICa-L) has been controversial and is the topic of this study. To determine if and how sustained increases in ICa-L induces PCH, transgenic mouse models with low (LE) and high (HE) expression levels of the β2a subunit of Ca2+ channels (β2a) and in cultured adult feline (AF) and neonatal rat (NR) ventricular myocytes (VMs) infected with an adenovirus containing a β2a-GFP.

Results

In vivo, β2a LE and HE mice had increased heart weight to body weight ratio, posterior wall and interventricular septal thickness, tissue fibrosis, myocyte volume and cross sectional area and the expression of PCH markers in a time- and dose- dependent manner. PCH was associated with a hypercontractile phenotype including enhanced ICa-L, fractional shortening, peak Ca2+ transient, at the myocyte level, greater ejection fraction and fractional shortening at the organ level. In addition, LE mice had an exaggerated hypertrophic response to transverse aortic constriction. In vitro overexpression of β2a in cultured AFVMs increased ICa-L, cell volume, protein synthesis, NFAT and HDAC translocations and in NRVMs increased surface area. These effects were abolished by the blockade of ICa-L, intracellular Ca2+, calcineurin, CaMK II and SERCA.

Conclusion

Increasing ICa-L is sufficient to induce PCH through the calcineurin/NFAT and CaMKII/HDAC pathways. Both cytosolic and SR/ER-nuclear envelop Ca2+ pools were shown to be involved.

Kiamycin (1), a new angucyclinone derivative possessing an 1,12-epoxybenz[a]anthracene ring system, was isolated from the marine Streptomyces sp. strain M268 along with the known compounds 8-O-methyltetrangomycin (3) and 8-O-methylrabelomycin (4). Their structures were elucidated by detailed spectroscopic analysis and comparison with literature data. The new angucyclinone derivative showed inhibitory activities against the human cell lines HL-60 (leukemia), A549 (lung adenocarcinoma), and BEL-7402 (hepatoma) with inhibition rates of 68.2%, 55.9%, and 31.7%, respectively, at 100 µM. It appears to have potential as an anticancer agent with selective activity.

In our previous study, we attempted to develop a tool for the early diagnosis of non-small cell lung cancer (NSCLC) using DNA methylation biomarkers. With the aim of improving the diagnostic potential by optimizing the composition of the target set, in this study, 13 candidate genes (ACTA1, AIDH1A2, CBX8, CDH8, EVX1, MGC16275, NEUROG1, NEUROG2, NID2, OTX2OS1, PGAM2, PHOX2B and TOX) were analyzed by methylation-specific PCR to determine the methylation status of each gene in 5 NSCLC cell lines and in lung tissue samples from 15 healthy volunteers, 103 stage I NSCLC patients and 26 non-cancerous control patients. Results showed that NEUROG2 and NID2 were hypermethylated in stage I NSCLC tissues (31.07 and 46.60%, respectively) and unmethylated in normal lung tissues (0/15) and non-cancerous tissues (0/26). Following recombination, an optimized 5-gene panel (NEUROG2, NID2, RASSF1A, APC and HOXC9) achieved a sensitivity of 91.26% with a specificity of 84.62% in the detection of stage I NSCLC. The optimized 5-gene panel greatly improved the diagnostic power for stage I NSCLC.

In this study, 207 strains of aerobic and facultatively anaerobic cellulolytic bacteria were isolated from the gut of Holotrichia parallela larvae. These bacterial isolates were assigned to 21 genotypes by amplified ribosomal DNA restriction analysis (ARDRA). A partial 16S rDNA sequence analysis and standard biochemical and physiological tests were used for the assignment of the 21 representative isolates. Our results show that the cellulolytic bacterial community is dominated by the Proteobacteria (70.05%), followed by the Actinobacteria (24.15%), the Firmicutes (4.35%), and the Bacteroidetes (1.45%). At the genus level, Gram-negative bacteria including Pseudomonas, Ochrobactrum, Rhizobium, Cellulosimicrobium, and Microbacterium were the predominant groups, but members of Bacillus, Dyadobacter, Siphonobacter, Paracoccus, Kaistia, Devosia, Labrys, Ensifer, Variovorax, Shinella, Citrobacter, and Stenotrophomonas were also found. Furthermore, our results suggest that a significant amount of bacterial diversity exists among the cellulolytic bacteria, and that Siphonobacter aquaeclarae, Cellulosimicrobium funkei, Paracoccus sulfuroxidans, Ochrobactrum cytisi, Ochrobactrum haematophilum, Kaistia adipata, Devosia riboflavina, Labrys neptuniae, Ensifer adhaerens, Shinella zoogloeoides, Citrobacter freundii, and Pseudomonas nitroreducens are reported to be cellulolytic for the first time in this study. Our results indicate that the scarab gut is an attractive source for the study of novel cellulolytic microorganisms and enzymes useful for cellulose degradation.

Our goal was to identify the bacteria inhabiting the reproductive system of the female oriental fruit fly, Bactrocera dorsalis (Hendel), and evaluate the chemotaxis of B. dorsalis to the metabolites produced by the bacteria. Based on 16S rRNA-based polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), 18 operational taxonomic units (OTUs) were assigned to the five bacterial classes Betaproteobacteria, Alphaproteobacteria, Gammaproteobacteria, Bacilli and Actinobacteria. Nine OTUs were assigned to Gammaproteobacteria, which was the most highly represented class. Enterobacteriaceae constituted the dominant family, and within this family, three genera and five species were identified, including Enterobacter sakazakii, Klebsiella oxytoca, Klebsiella pneumoniae, Raoultella terrigena and Enterobacter amnigenus. In this set, the first two species were the dominant components, and the latter three species were the minor ones. Finally, we found that the metabolites produced by R. terrigena, K. oxytoca and K. pneumoniae were attractive to the B. dorsalis adults, and in field studies, B. dorsalis adults were most attracted to K. oxytoca. Collectively, our results suggest that the female reproductive system plays an important role in the transfer of enterobacteria from the gut to fruit. Our data may prompt the development of a female-targeted population control strategy for this fly.

Cisplatin resistance is one of the major reasons leading to the high death rate of ovarian cancer. Methyl-Capture sequencing (MethylCap-seq), which combines precipitation of methylated DNA by recombinant methyl-CpG binding domain of MBD2 protein with NGS, global and unbiased analysis of global DNA methylation patterns. We applied MethylCap-seq to analyze genome-wide DNA methylation profile of cisplatin sensitive ovarian cancer cell line A2780 and its isogenic derivative resistant line A2780CP. We obtained 21,763,035 raw reads for the drug resistant cell line A2780CP and 18,821,061reads for the sensitive cell line A2780. We identified 1224 hyper-methylated and 1216 hypomethylated DMRs (differentially methylated region) in A2780CP compared to A2780. Our MethylCap-seq data on this ovarian cancer cisplatin resistant model provided a good resource for the research community. We also found that A2780CP, compared to A2780, has lower observed to expected methylated CpG ratios, suggesting a lower global CpG methylation in A2780CP cells. Methylation specific PCR and bisulfite sequencing confirmed hypermethylation of PTK6, PRKCE and BCL2L1 in A2780 compared with A2780CP. Furthermore, treatment with the demethylation reagent 5-aza-dC in A2780 cells demethylated the promoters and restored the expression of PTK6, PRKCE and BCL2L1.

Antagonists of L-type Ca2+ channels (LTCCs) have been used to treat human cardiovascular diseases for decades. However, these inhibitors can have untoward effects in patients with heart failure, and their overall therapeutic profile remains nebulous given differential effects in the vasculature when compared with those in cardiomyocytes. To investigate this issue, we examined mice heterozygous for the gene encoding the pore-forming subunit of LTCC (calcium channel, voltage-dependent, L type, α1C subunit [Cacna1c mice; referred to herein as α1C–/+ mice]) and mice in which this gene was loxP targeted to achieve graded heart-specific gene deletion (termed herein α1C-loxP mice). Adult cardiomyocytes from the hearts of α1C–/+ mice at 10 weeks of age showed a decrease in LTCC current and a modest decrease in cardiac function, which we initially hypothesized would be cardioprotective. However, α1C–/+ mice subjected to pressure overload stimulation, isoproterenol infusion, and swimming showed greater cardiac hypertrophy, greater reductions in ventricular performance, and greater ventricular dilation than α1C+/+ controls. The same detrimental effects were observed in α1C-loxP animals with a cardiomyocyte-specific deletion of one allele. More severe reductions in α1C protein levels with combinatorial deleted alleles produced spontaneous cardiac hypertrophy before 3 months of age, with early adulthood lethality. Mechanistically, our data suggest that a reduction in LTCC current leads to neuroendocrine stress, with sensitized and leaky sarcoplasmic reticulum Ca2+ release as a compensatory mechanism to preserve contractility. This state results in calcineurin/nuclear factor of activated T cells signaling that promotes hypertrophy and disease.

Preclinical modeling of Parkinson's disease using 6-hydroxydopamine (6-OHDA) has been valuable in developing and testing therapeutic strategies. Recent efforts have focused on modeling early stages of disease by infusing 6-OHDA into the striatum. The partial DA depletion that follows intrastriatal 6-OHDA is more variable than the near complete depletion following medial forebrain bundle infusion, and behavioral screening assays are not as well characterized in the partial lesion model. We compared relationships between amphetamine-elicited rotation behavior and DA depletion following intrastriatal 6-OHDA (12.5 μg) in 6 month vs. 18 month F344/BN rats, at 2-weeks and 6-weeks post-lesion. We compared the total number of rotations with within-session (bin-by-bin) parameters of rotation behavior as indicators of DA depletion. Striatal DA depletion was greater in the young adult than in the middle-aged rats at 2 weeks but not at 6 weeks post-lesion. The total number of rotations for the whole session and striatal DA depletion did not differ between the two age groups. Regression analysis revealed a greater relationship between within-session parameters of rotation behavior and DA depletion in the middle-aged group than in the young adult group. These results have implications for estimating DA depletion in preclinical studies using rats of different ages.

Rationale

Myocardial infarction (MI) leads to heart failure (HF) and premature death. The respective roles of myocyte death and depressed myocyte contractility in the induction of HF after MI have not been clearly defined and are the focus of this study.

Objectives

We developed a mouse model in which we could prevent depressed myocyte contractility after MI and used it to test the idea that preventing depression of myocyte Ca2+-handling defects could avert post-MI cardiac pump dysfunction.

Methods and Results

MI was produced in mice with inducible, cardiac-specific expression of the β2a subunit of the L-type Ca2+ channel. Myocyte and cardiac function were compared in control and β2a animals before and after MI. β2a myocytes had increased Ca2+ current; sarcoplasmic reticulum Ca2+ load, contraction and Ca2+ transients (versus controls), and β2a hearts had increased performance before MI. After MI, cardiac function decreased. However, ventricular dilation, myocyte hypertrophy and death, and depressed cardiac pump function were greater in β2a versus control hearts after MI. β2a animals also had poorer survival after MI. Myocytes isolated from β2a hearts after MI did not develop depressed Ca2+ handling, and Ca2+ current, contractions, and Ca2+ transients were still above control levels (before MI).

Conclusions

Maintaining myocyte contractility after MI, by increasing Ca2+ influx, depresses rather than improves cardiac pump function after MI by reducing myocyte number.

In order to avoid low absorption, incorporation, and undesirable side effects of inorganic oxovanadium compounds, the antidiabetic activities of organic oxovanadium (IV) compounds in alloxan-induced diabetic mice were investigated. Vanadyl carboxymethyl carrageenan (VOCCA) and vanadyl carboxymethyl chitosan (VOCCH) were synthesized and administrated through intragastric administration in different doses for 20 days in alloxan-induced diabetic mice. Glibenclamide was administrated as the positive control. Our results showed that low-dose group, middle-dose group, and high-dose group of VOCCA and VOCCH could significantly reduce the levels of blood glucose (P < 0.05) compared with untreated group, but not in normal mice. Besides, high-dose groups of VOCCA and VOCCH exhibited more significant hypoglycemic activities (P < 0.01). After treated with VOCCH, the oral glucose tolerance of high-dose group of VOCCH was improved compared with model control group (P < 0.05).

Genomic imprinting causes the expression of an allele depending on its parental origin. In plants, most imprinted genes have been identified in Arabidopsis endosperm, a transient structure consumed by the embryo during seed formation. We identified imprinted genes in rice seed where both the endosperm and embryo are present at seed maturity. RNA was extracted from embryos and endosperm of seeds obtained from reciprocal crosses between two subspecies Nipponbare (Japonica rice) and 93-11 (Indica rice). Sequenced reads from cDNA libraries were aligned to their respective parental genomes using single-nucleotide polymorphisms (SNPs). Reads across SNPs enabled derivation of parental expression bias ratios. A continuum of parental expression bias states was observed. Statistical analyses indicated 262 candidate imprinted loci in the endosperm and three in the embryo (168 genic and 97 non-genic). Fifty-six of the 67 loci investigated were confirmed to be imprinted in the seed. Imprinted loci are not clustered in the rice genome as found in mammals. All of these imprinted loci were expressed in the endosperm, and one of these was also imprinted in the embryo, confirming that in both rice and Arabidopsis imprinted expression is primarily confined to the endosperm. Some rice imprinted genes were also expressed in vegetative tissues, indicating that they have additional roles in plant growth. Comparison of candidate imprinted genes found in rice with imprinted candidate loci obtained from genome-wide surveys of imprinted genes in Arabidopsis to date shows a low degree of conservation, suggesting that imprinting has evolved independently in eudicots and monocots.

Author Summary

The expression of maternal or paternal alleles in either a preferentially or exclusively uniparental manner, termed imprinting, is prevalent in the transient endosperm of seeds in the model plant Arabidopsis. Cereals form seeds where both the embryo and endosperm are present at seed maturity. They are an important world food source. To date, very few imprinted genes have been identified in cereal seeds. How parental gene expression biases contribute to rice seed development has not yet been studied in detail. The deep resolution of transcript sequencing platforms was used to identify loci expressed in a parentally biased manner in the embryo and endosperm of Indica and Japonica rice at a genome-wide level. We identified 262 candidate imprinted loci expressed in the endosperm, experimentally verified 56 of these, and found novel features pertaining to their expression. Only one gene was found to be imprinted in the rice embryo. Imprinting in Arabidopsis and rice seeds is confined primarily to the endosperm, but the identified loci do not share extensive sequence conservation. Imprinting thus appears to have evolved independently in these plant species.