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Suppression of the cardiac sodium channel NaV1.5 leads to fatal arrhythmias in ischemic heart disease (IHD). However, the transcriptional regulation of NaV1.5 in cardiac ischemia is still unclear. Our studies are aimed to investigate the expression of enhancer of zeste homolog 2 (EZH2) in IHD and regulation of cardiac NaV1.5 expression by EZH2. Human heart tissue was obtained from IHD and non-failing heart (NFH) patients; mouse heart tissue was obtained from the peri-infarct zone of hearts with myocardial infarction (MI) and hearts with a sham procedure. Protein and mRNA expression were measured by immunoblotting, immunostaining, and qRT-PCR. Protein-DNA binding and promoter activity were analyzed by ChIP-qPCR and luciferase assays, respectively. Na+ channel activity was assessed by whole-cell patch clamp recordings. EZH2 and H3K27me3 were increased while NaV1.5 expression was reduced in IHD hearts and in mouse MI hearts compared to the controls. Reduced NaV1.5 and increased EZH2 mRNA levels were observed in mouse MI hearts. A selective EZH2 inhibitor, GSK126 decreased H3K27me3 and elevated NaV1.5 in HL-1 cells. Silencing of EZH2 expression decreased H3K27me3 and increased NaV1.5 in these cells. EZH2 and H3K27me3 were enriched in the promoter regions of Scn5a and were decreased by treatment with EZH2 siRNA. GSK126 inhibited the enrichment of H3K27me3 in the Scn5a promoter and enhanced Scn5a transcriptional activity. GSK126 significantly increased Na+ channel activity. Taken together, EZH2 is increased in ischemic hearts and epigenetically suppresses Scn5a transcription by H3K27me3, leading to decreased NaV1.5 expression and Na+ channel activity underlying the pathogenesis of arrhythmias.

Mutations in kinases are abundant and critical to study signaling pathways and regulatory roles in human disease, especially in cancer. Somatic mutations in kinase genes can affect drug treatment, both sensitivity and resistance, to clinically used kinase inhibitors. Here, we present a newly constructed database, KinaseMD (kinase mutations and drug response), to structurally and functionally annotate kinase mutations. KinaseMD integrates 679 374 somatic mutations, 251 522 network-rewiring events, and 390 460 drug response records curated from various sources for 547 kinases. We uniquely annotate the mutations and kinase inhibitor response in four types of protein substructures (gatekeeper, A-loop, G-loop and αC-helix) that are linked to kinase inhibitor resistance in literature. In addition, we annotate functional mutations that may rewire kinase regulatory network and report four phosphorylation signals (gain, loss, up-regulation and down-regulation). Overall, KinaseMD provides the most updated information on mutations, unique annotations of drug response especially drug resistance and functional sites of kinases. KinaseMD is accessible at https://bioinfo.uth.edu/kmd/, having functions for searching, browsing and downloading data. To our knowledge, there has been no systematic annotation of these structural mutations linking to kinase inhibitor response. In summary, KinaseMD is a centralized database for kinase mutations and drug response.

Background β-Catenin/T-cell factor 4 (TCF4) signaling is enhanced in ischemic heart disease in which ventricular tachycardia (VT)/ventricular fibrillation occurs frequently. How this signaling links to arrhythmogenesis remains unclear. Objective The purpose of this study was to investigate the role of β-catenin gain of function in the development of arrhythmia. Methods A mouse model with a conditional deletion of CTNNB1 exon 3 resulting in cardiac exon 3–deleted and stabilized β-catenin (β-catΔE3) was used to determine the role of β-catenin gain of function in the regulation of cardiac rhythm. Results Western blotting showed β-catΔE3 expression and significantly decreased NaV1.5 protein in CTNNB1 E3−/− and CTNNB1 E3+/− mouse hearts. Real-time qRT-PCR revealed significantly decreased NaV1.5 messenger RNA with no changes in Na+ channel β1 to β4 expression in these hearts. Immunofluorescence revealed accumulation of β-catΔE3 in the nuclei of CTNNB1 E3−/− cardiomyocytes. Immunohistochemistry demonstrated nuclear localization of β-catenin in cardiomyocytes, which was associated with significantly decreased NaV1.5 messenger RNA in human ischemic hearts. Immunoprecipitation revealed that β-catΔE3 interacted with TCF4 in CTNNB1 E3−/− cardiomyocytes. Whole-cell recordings showed that Na+ currents and depolarization and amplitude of action potentials were significantly decreased in CTNNB1 E3−/− ventricular myocytes. Electrocardiographic recordings demonstrated that in mice with cardiac CTNNB1 E3−/−, the QRS complex was prolonged and VT was induced by the Na+ channel blocker flecainide. However, cardiac function, as determined by echocardiography and heart/body weight ratios, remained unchanged. Conclusion Enhancement of β-catenin/TCF4 signaling led to the prolongation of the QRS complex and increase in susceptibility to VT by suppression of NaV1.5 expression and Na+ channel activity in mice.

Increased nuclear β-catenin interacting with T-cell factor 4 (TCF4) affects the expression of target genes including SCN5A in ischemic heart disease, which is characterized by frequent ventricular tachycardia/fibrillation. A complex of β-catenin and TCF4 inhibits cardiac Na+ channel activity by reducing NaV1.5 expression through suppressing SCN5A promoter activity in HL-1 cardiomyocytes. LF3, a 4-thioureido-benzenesulfonamide derivative and an inhibitor of β-catenin/TCF4 interaction, has been shown to block the self-renewal capacity of cancer stem cells. We performed studies to determine if LF3 can reverse suppressive effects of β-catenin/TCF4 signaling on the expression of NaV1.5 in HL-1 cardiomyocytes. Western blotting and real-time qRT-PCR analyses showed that 10 μM LF3 significantly increased the expression of NaV1.5 but it did not alter β-catenin and TCF4 expression. Subcellular fractionation analysis demonstrated that LF3 significantly increased the levels of NaV1.5 in both membrane and cytoplasm. Whole-cell patch-clamp recordings revealed that Na+ currents were significantly increased with no changes in the steady-state parameters, activation and inactivation time constants and recovery from inactivation of Na+ channel in HL-1 cells treated with LF3. Immunoprecipitation exhibited that LF3 blocked the interaction of β-catenin and TCF4. Luciferase reporter assays performed in HEK 293 cells and HL-1 revealed that LF3 increased the SCN5A promoter activity in HL-1 cells and prevented β-catenin suppressive effect on SCN5A promoter activity in HEK 293 cells. Taken together, we conclude that LF3, an inhibitor of β-catenin/TCF4 interaction, elevates NaV1.5 expression, leading to increase Na+ channel activity in HL-1 cardiomyocytes.

Approximately 15% of human cancers are estimated to be attributed to viruses. Virus sequences can be integrated into the host genome, leading to genomic instability and carcinogenesis. Here, a new deep convolutional neural network (CNN) model is developed with attention architecture, namely DeepVISP, for accurately predicting oncogenic virus integration sites (VISs) in the human genome. Using the curated benchmark integration data of three viruses, hepatitis B virus (HBV), human herpesvirus (HPV), and Epstein‐Barr virus (EBV), DeepVISP achieves high accuracy and robust performance for all three viruses through automatically learning informative features and essential genomic positions only from the DNA sequences. In comparison, DeepVISP outperforms conventional machine learning methods by 8.43–34.33% measured by area under curve (AUC) value enhancement in three viruses. Moreover, DeepVISP can decode cis‐regulatory factors that are potentially involved in virus integration and tumorigenesis, such as HOXB7, IKZF1, and LHX6. These findings are supported by multiple lines of evidence in literature. The clustering analysis of the informative motifs reveales that the representative k‐mers in clusters could help guide virus recognition of the host genes. A user‐friendly web server is developed for predicting putative oncogenic VISs in the human genome using DeepVISP., Approximately 15% of human cancers are estimated to be attributed to viruses. This study presents a new deep learning model with attention architecture, namely DeepVISP, for accurately predicting oncogenic virus integration sites (VISs) in the human genome. Moreover, DeepVISP decodes several cis‐regulatory factors that are potentially involved in virus integration and tumorigenesis.

Bone morphogenetic protein (Bmp) and Wnt pathways regulate cell proliferation and differentiation, but how these two pathways interact and mediate their nuclear actions in the heart, especially during late cardiac development, remains poorly defined. T-cell factor (TCF) and lymphoid enhancer factor (LEF) family transcriptional factors, including Lef1, Tcf7, Tcf7l1, and Tcf7l2, are important nuclear mediators of canonical Wnt/β-catenin signaling throughout cardiac development. We reveal that these TCF/LEF family members direct heart maturation through distinct temporal and spatial control. Tcf7 and Lef1 decreases while Tcf7l1 and Tcf7l2 remain relatively stable during heart development. Lef1 is mainly expressed in mesenchymal cells in valvular regions. Tcf7 and Tcf7l1 are detected in the nucleus of mesothelial and endothelial cells, but not in cardiomyocytes or mesenchymal cells. Tcf7l2 is the primary TCF/LEF family member in cardiomyocytes and undergoes alternative splicing during heart development. A Tcf7l2 intensity gradient opposite to that of β-catenin and cardiomyocyte proliferative activity is present in fetal hearts. Wnt activation by cardiac deletion of adenomatosis polyposis coli, a negative Wnt regulator, dramatically increases Cyclin D2 and Bmp4 expression. Bmp signal transducing transcription factors, the mothers against decapentaplegic homologs (Smads) are increasingly phosphorylated upon Wnt activation. Lef1/Tcf7 displaces Tcf7l2 and cooperates with pSmad1/5/8 in the regulatory elements of Cyclin D2 and Bmp4 promoters to promote β-catenin recruitment and transcriptional activation. Finally, we demonstrate that Tcf7l2 is a transcriptional suppressor of Cyclin D2 and Bmp 4 in a cardiac cell line by overexpression and knockdown experiments.

High levels of glutamate dehydrogenase (GDH) activity are associated with hypoglycemia, cancer, and Parkinson's disease. Propylselen was synthesized to investigate its mechanism of GDH inhibition in comparison with Ebselen and Epigallocatechin gallate (EGCG). Because Ebselen was found to crosslink with the peptide (AA299-341) at the active site of E.coli GDH, the Cys, Pro, and Lys residues of the corresponding peptide were mutagenized to Ala residues. Using enzyme kinetics and biomolecular interaction assays, we found that the conserved GDH P320 residue is important for propylselen binding, C321 for Ebselen binding, and K341 for EGCG binding. In addition, these 3 mutations abolished NADP+ binding to E. coli GDH in the absence of glutamate substrate, but in the presence of glutamate, the catalytic activity of the mutants was reduced only by 2–4 fold, indicating that a substrate-induced fit mechanism exists in E. coli GDH. Furthermore, biochemical analysis showed that NADP+ had high affinity (Kd of 77 nM) for GDH; by targeting the NADP binding site, propylselen effectively inhibited both E. coli and human GDH activity and improved anticancer activity.

The BIG Data Center at Beijing Institute of Genomics (BIG) of the Chinese Academy of Sciences provides a suite of database resources in support of worldwide research activities in both academia and industry. With the vast amounts of multi-omics data generated at unprecedented scales and rates, the BIG Data Center is continually expanding, updating and enriching its core database resources through big data integration and value-added curation. Resources with significant updates in the past year include BioProject (a biological project library), BioSample (a biological sample library), Genome Sequence Archive (GSA, a data repository for archiving raw sequence reads), Genome Warehouse (GWH, a centralized resource housing genome-scale data), Genome Variation Map (GVM, a public repository of genome variations), Science Wikis (a catalog of biological knowledge wikis for community annotations) and IC4R (Information Commons for Rice). Newly released resources include EWAS Atlas (a knowledgebase of epigenome-wide association studies), iDog (an integrated omics data resource for dog) and RNA editing resources (for editome-disease associations and plant RNA editosome, respectively). To promote biodiversity and health big data sharing around the world, the Open Biodiversity and Health Big Data (BHBD) initiative is introduced. All of these resources are publicly accessible at http://bigd.big.ac.cn.

The bone morphogenetic protein (BMP) signaling pathway is highly conserved across many species, and its importance for the patterning of the skeletal system has been demonstrated. A disrupted BMP signaling pathway results in severe skeletal defects. Murine calvaria has been identified to have dual-tissue lineages, namely, the cranial neural-crest cells and the paraxial mesoderm. Modulations of the BMP signaling pathway have been demonstrated to be significant in determining calvarial osteogenic potentials and ossification in vitro and in vivo. More importantly, the BMP signaling pathway plays a role in the maintenance of the homeostasis of the calvarial stem cells, indicating a potential clinic significance in calvarial bone and in expediting regeneration. Following the inherent evidence of BMP signaling in craniofacial biology, we summarize recent discoveries relating to BMP signaling in the development of calvarial structures, functions of the suture stem cells and their niche and regeneration. This review will not only provide a better understanding of BMP signaling in cranial biology, but also exhibit the molecular targets of BMP signaling that possess clinical potential for tissue regeneration.

Histopathologic diagnosis of antibody-mediated rejection in posttransplant liver biopsies is challenging. The recently proposed diagnostic criteria by the Banff Working Group on Liver Allograft Pathology require positive C4d immunohistochemical staining to establish the diagnosis. However, the reported C4d staining patterns vary widely in different studies. One potential explanation may be due to different antibody preparations used by different investigators. In this study, posttransplant liver biopsies from 69 patients histopathologically diagnosed with acute cellular rejection, chronic rejection, or recurrent hepatitis C were immunohistochemically stained using 2 polyclonal anti-C4d antibodies. On the basis of the distribution of C4d immunoreactivity, 5 different staining patterns were observed: portal vein and capillary, hepatic artery, portal stroma, central vein, and sinusoids. The frequency, extent, and intensity of positive C4d staining with the 2 antibody preparations differed significantly for portal veins/capillaries and central veins, but not for hepatic arteries and portal stroma. Positive sinusoidal staining was seen in only 1 case. There were no significant differences in the frequency, extent, and intensity of positive C4d staining among the acute cellular rejection, chronic rejection, and recurrent hepatitis C groups with the 2 anti-C4d antibodies. These data show that different anti-C4d antibodies can show different staining patterns, which may lead to different interpretation. Caution is thus needed when selecting C4d antibodies for clinical use to aid in the diagnosis of antibody-mediated rejection.

Genetic manipulation of key components of the evolutionally conserved Hippo pathway has shown that the precise control of these signaling molecules is critical to cardiac development and response to stresses. However, how this pathway is involved in the progression of cardiac dysfunction in different heart diseases remains unclear. We investigated the expressional levels and subcellular localization of Yap1, Taz, and Tead1 and determined Hippo target gene expression in failing human hearts with ischemic heart disease (IHD) and idiopathic dilated cardiomyopathy (IDC) and mouse desmin-related cardiomyopathy (DES). Our results demonstrated that Yap1, Taz, and Tead1 were significantly increased in failing human and DES hearts compared with the non-failing controls (NFH) or wild type (WT) mouse hearts at both mRNA and protein levels. Interestingly, adult human and mouse hearts had more Taz than Yap1 by mRNA and protein expression and their increases in diseased hearts were proportional and did not change Yap1/Taz ratio. Yap1, Taz, and Tead1 were accumulated in the nuclear fraction and cardiomyocyte nuclei of diseased hearts. The ratio of Yap1 phosphorylated at serine 127 (human) or serine 112 (mouse) to the total Yap1 (pYap1/Yap1) was significantly lower in the nuclear fraction of diseased hearts than that in normal controls. More importantly, Hippo downstream targets Ankrd1, Ctgf, and Cyr61 were transcriptionally elevated in the diseased hearts. These results suggest that Yap1/Taz signaling is activated in human and mouse dysfunctional hearts. Further investigation with relevant animal models will determine whether this pathway is a potential target for preventing and reversing abnormal remodeling during the progression of different cardiac disorders.

Oxidants and canonical Wnt/β-catenin signaling have been shown to decrease cardiac Na(+) channel activity by suppressing NaV1.5 expression. Our aims are to determine if hydrogen peroxide (H2O2), one oxidant of reactive oxygen species (ROS), activates Wnt/β-catenin signaling and promotes β-catenin nuclear activity, leading to suppression of NaV1.5 expression and if this suppression requires the interaction of β-catenin with its nuclear partner, TCF4 (also called TCF7L2) to decrease SCN5a promoter activity. The results demonstrated that H2O2 increased β-catenin, but not TCF4 nuclear localization determined by immunofluorescence without affecting total β-catenin protein level. Furthermore, H2O2 exerted a dose- and time-dependent suppressive effect on NaV1.5 expression. RT-PCR and/or Western blot analyses revealed that overexpressing active form of β-catenin or stabilizing β-catenin by GSK-3β inhibitors, LiCl and Bio, suppressed NaV1.5 expression in HL-1 cells. In contrast, destabilization of β-catenin by a constitutively active GSK-3β mutant (S9A) upregulated NaV1.5 expression. Whole-cell recording showed that LiCl significantly inhibited Na(+) channel activity in these cells. Using immunoprecipitation (IP), we showed that β-catenin interacted with TCF4 indicating that β-catenin as a co-transfactor, regulates NaV1.5 expression through TCF4. Analyses of the SCN5a promoter sequences among different species by using VISTA tools indicated that SCN5a promoter harbors TCF4 binding sites. Chromatin IP assays demonstrated that both β-catenin and TCF4 were recruited in the SCN5a promoter, and regulated its activity. Luciferase promoter assays exhibited that β-catenin inhibited the SCN5a promoter activity at a dose-dependent manner and this inhibition required TCF4. Small interfering (Si) RNA targeting β-catenin significantly increased SCN5a promoter activity, leading to enhanced NaV1.5 expression. As expected, β-catenin SiRNA prevents H2O2 suppressive effects on both SCN5a promoter activity and NaV1.5 expression. Our findings indicate that H2O2 inhibits NaV1.5 expression by activating the Wnt/β-catenin signaling and β-catenin interacts with TCF4 to transcriptionally suppress cardiac NaV1.5 expression.

DNA double strand breaks (DSBs) are most dangerous lesions. To determine whether oxidative stress can induce DSBs and how they are repaired in cardiomyocytes (CMs), cultured neonatal rat CMs were treated with different doses of H2O2 and followed for up to 72 hours for monitoring the spatiotemporal dynamics of DNA repair protein assembly/disassembly at DSB foci. The protein levels and foci numbers of histone H2AX phosphorylated at serine 139 (γ-H2AX) increased proportionally to 50, 100 and 200 µmol/L H2O2 after 30 min treatment. When H2O2 was at or above 400 µmol/L, γ-H2AX became predominantly pan-nuclear. After 30 min, 200 µmol/L of H2O2 treatment, γ-H2AX levels were highest within the first hour and then gradually declined during the recovery and returned to basal levels at 48 hours. Among DNA damage transducer kinases, ataxia telangiectasia mutated (ATM) was significantly activated by H2O2 in contrast to mild activation of ataxia telangiectasia mutated and Rad3-related (ATR). DSB binding protein, p53 binding protein 1 formed distinct nuclear foci that colocalized with γ-H2AX foci and phosphorylated ATM. Our findings indicate that DNA double strand breaks can be induced by H2O2 and ATM is the main kinase to mediate DSB repair in cardiomyocytes. Therefore, monitoring DSB repair can assess oxidative injury and response in cardiomyocytes.

Mammalian/human achaete-scute homolog 1 (hASH1) regulates neuroendocrine cell development. No detailed comparative study has been conducted to explore the immunohistochemical utility of hASH1 in distinguishing different types of lung cancers. We investigated the expression of hASH1, synaptophysin, chromogranin, and CD56 in 101 squamous cell carcinomas (SCCs), 183 adenocarcinomas (ADCs), 37 typical carcinoids (TCs), 14 atypical carcinoids (ACs), 11 large cell neuroendocrine carcinomas (LCNECs), and 24 small cell lung carcinomas (SCLCs) of the lung by immunohistochemical staining with a monoclonal antibody against hASH1. Staining intensity was graded from 0 to 3, and percentage of tumor cells in each grade was estimated. All cases of ADC and SCC were discreetly negative for hASH1 in contrast to their low percentage positivity for synaptophysin, chromogranin, and CD56. hASH1 positively stained TC (64.9%), AC (64.3%), LCNEC (72.7%), and SCLC (79.2%) as did chromogranin (TC, 100%; AC, 78.6%; LCNEC, 9.0%; and SCLC, 4.2%), synaptophysin (TC, 100%; AC, 78.6%; LCNEC, 81.8%; and SCLC, 83.3%), and CD56 (TC, 59.5%; AC, 57.1%; LCNEC, 36.4%; and SCLC, 79.2%). TC and AC often showed weaker intensity and lower percentage of tumor cells positive for hASH1 (median score, 5) than LCNEC and SCLC (median score, 40 and 170, respectively). There were statistically significant differences in mean intensity scores between SCLC (148.8 ± 20.1) and other neuroendocrine tumors: TC (37.1 ± 9.2) and AC (28.6 ± 10.8) or LCNEC (51.8 ± 18.0). Our findings indicate that hASH1 is a specific marker to distinguish neuroendocrine tumors from SCC and ADC. Additionally, hASH1 is a useful diagnostic marker for segregating SCLC from other neuroendocrine tumors.

Aims Cardiomyocyte (CM) proliferation increases from the inner trabecular to outer compact myocardium in fetal hearts. We determined if canonical Wnt signaling has directional and graded activity to maintain this CM proliferation gradient. Moreover, we investigated whether perturbation of Wnt signaling intensity could modulate CM proliferative activity. Methods and results With confocal microscopy and image analysis we found that the Wnt effector, β-catenin, formed a signaling gradient which positively correlated with CM proliferative activity across ventricular walls of wild type (WT) embryos at embryonic day (E) 13.5 and 17.5. Negative Wnt regulators, adenomatosis polyposis coli (APC), had a reverse distribution pattern. The activation of canonical Wnt/β-catenin signaling by deletion of Apc in CMs led to ventricular hyperplasia with no adverse effects on fetal survival or CM differentiation. In contrast, cardiac deletion of β-catenin resulted in ventricular hypoplasia and fetal demise by E14.5. We further revealed differential distribution and regulation of three cyclin Ds in fetal hearts. Cyclin D1 was mainly expressed in endothelial cells. Although both cyclin D2 and D3 were present in CMs, only cyclin D2 was regulated by Wnt signaling perturbation: downregulation by β-catenin deletion and upregulation by Apc knockout. Conclusion Canonical Wnt signaling is asymmetrical and graded across ventricular walls and positively regulates CM proliferation via cyclin D2.

Background: Pulmonary sclerosing pneumocytoma (PSP) typically presents solitary and peripheral mass, while only rarely cases display unusual multiple lesions. We reported a extremely rare case of PSP with diffusely-scattered nodules in the right lung. Case presentation: Diffusely round-shaped nodular shadows in the right lung were found by CT scan in a 31-year-old Chinese woman. The patient undergone the right pneumnectomy. Grossly, numerous small nodules, up to 2.5 cm in greatest dimension were identified in the upper, middle and lower lobes of the right lung. Histologically, the tumor presented the typical features of PSP, with a variable proportion of solid, sclerotic and papillary patterns. Immunohistochemical staining further revealed that cuboidal surface epithelial cells were positive for TTF-1, EMA, AE1/3 and vimentin (partially), and round or polygonal cells expressed TTF-1, vimentin, EMA (weakly), synaptophysin (partially), progesterone receptor (partially), and estrogen receptor (scatteredly). The patient has been followed up for 83 months after surgery by annual chest CT and no new lesions are detected in her left lung and other organs. The whole-exome sequencing identified 15 somatic mutations genes (MEGF6, DNAH5, AKT1, GPRIN2, PIK3AP1, FBXO40, HERC1, VPS16, MORN1, ZNF474, CTNNB1, ZNF251, TSC1, ATM, KDR). Pathway analysis showed possible pathways like the components of CTNNB1, AKT1, and TSC1 mutations in the PI3K/AKT signalings and AKT1, KDR and ATM in VEGF signaling pathway and AKT1 activation seemed closely related with these pathways. Conclusion: According to our and previous data, PSP with diffuse or multiple lesions is very rare, and the patients are most commonly seen in women in Asian countries. The misdiagnosis rate by clinical and intraoperative frozen-section assessment is high because of the multiple nodules in the lung and its confusing histological features. Long time follow up indicates surgical resection should not be considered as the preferred strategy for treating multiple PSP in the intralobar sites. AKT1 activation may contribute to the development of PSP while the pathogenesis of diffuse or multiple PSP still needs to be further analyzed.

The human cardiac fast transient outward K+ channel is composed of the KV4.3 α subunit encoded by KCND3 and the K+ channel‑interacting protein 2 (KChIP2) β subunit, and determines the early repolarization of the action potential (AP). Two human mutations (G600R and L450F) in KV4.3 are associated with Brugada syndrome and they increase the KV4.3/KChIP2‑encoded fast transient outward K+ current (Ito,f) and cause the stable loss of the AP dome. However, the detailed mechanisms underlying the gain of Ito,f function by these two mutations are largely unknown. The experiments in the present study were undertaken to investigate the effect of these mutations and the underlying mechanism. Whole cell patch‑clamp recording was performed in HEK‑293 cells expressing KV4.3‑wild‑type (WT) and KV4.3 mutants with KChIP2. The two individual mutant‑encoded currents were significantly increased but the kinetics of the channels affected by the two mutations were different. The two mutations slowed KV4.3/KChIP2‑encoded channel inactivation; they did not increase the recovery from the KV4.3/KChIP2‑encoded channel inactivation. Western blotting showed that total KV4.3 protein was significantly augmented in HEK‑293 cells expressing the two individual mutants with KChIP2. Furthermore, immunofluorescence confocal microscopy demonstrated that the KV4.3 channel protein was expressed more in the cell membrane compared to the cytoplasm in cells that expressed individual mutants with KChIP2. Also, KChIP2 increased the amount of channel protein in the cell membrane of KV4.3 mutants significantly more than KV4.3‑WT. Reverse transcription‑polymerase chain reaction showed that KV4.3 mRNA was not significantly changed by individual mutations in the presence of KChIP2. Taken together, the present study revealed that the mutations cause a gain‑of‑function of KV4.3/KChIP2‑encoded channels by increasing membrane protein expression and slowing channel inactivation.

Introduction: In vitro studies showed that activation of β-catenin suppresses Na V 1.5 expression by inhibiting SCN5a promoter activity, leading to a decrease of Na + channel activity. How β-catenin regulates cardiac electrophysiological phenotype is unknown. Hypothesis: We hypothesized that cardiac activation of β-catenin regulates electrophysiological phenotype by suppressing Na V 1.5 expression. Methods: Adult mice with cardiac-specific, tamoxifen-induced deletion of β-catenin exon3 , leading to cardiac activation of β-catenin (β-catenin exon3 -/- ) were generated, and the effects of cardiac activation of β-catenin on the electrophysiological remodeling were assessed by electrocardiogram (ECG) recording. Class Ic antiarrhythmic reagent, flecainide, was administered to evaluate susceptibility to ventricular tachycardia (VT). Cardiac structure and function were evaluated by histologic and echocardiographic examinations, respectively. Western blot and qRT-PCR were performed to determine the levels of Na V 1.5 and β-catenin expression in mouse hearts. Whole-cell recording technique was utilized to record Na + currents and action potentials (APs) from ventricular myocytes. Results: Histologic and echocardiographic examinations showed that β-catenin exon3 -/- mice had normal cardiac structure and function. Compared to wild type (WT) mice, the ratio of heart/body weight was not changed and the duration of QRS was significantly prolonged in β-catenin exon3 -/- mice. VT was induced by flecainide in 60% of β-catenin exon3 -/- mice but not in WT mice. Western blot and qRT-PCR showed that Na V 1.5 protein and mRNA were significantly decreased in β-catenin exon3 -/- hearts, compared to WT hearts. Maximal upstroke velocity and amplitude of APs and Na + currents were significantly decreased in β-catenin exon3 -/- ventricular myocytes, compared to WT cells. Conclusion: Cardiac activation of β-catenin leads to prolongation of QRS and susceptibility to VT by decreasing Na V 1.5 expression and Na + channel activity.

Idiopathic interstitial pneumonias (IIPs) are a group of diffuse parenchymal lung diseases of unknown etiology characterized by the presence of various degrees of inflammation and fibrosis. We aimed to screen the differences among IIPs subtypes in the gene level by using the microarray expression profiles of normal lung tissue and IIPs tissue for the key genes associated with early diagnosis and treatment of IIPs.The gene expression profile of six kinds of IIPs (GSE 32537) subtypes tissue and normal lung tissues were downloaded. The differentially expressed genes (DEGs) in different IIPs subtypes were selected by using the expression profiling. In addition, the screened DEGs were further analyzed by function annotation, pathway analysis, and interaction network analysis to reveal the differences among these subtypes.The gene expression analysis showed that nine genes including SERPINA3, IL1R2, CBS, MGAM, SLCO4A1, S100A12, FPR1, SDR16C5, and MT1X in six subtypes of IIPs were significantly increased. There were significant differences in DEGs among six subtypes of IIPs, and the DEGs of some IIPs subtypes involved in immune, inflammatory response and cell adhesion processes. Moreover, the PPI network analysis indicated that SERPINA3 played an important role in the molecular mechanisms of IIPs.This comprehensive description of altered gene expression in different subtypes of IIPs underscores the complex biological processes characteristic of different subtypes of IIPs and may provide a foundation for future research into this devastating disease.

Mutation analysis of epidermal growth factor receptor (EGFR) is essential in determining the therapeutic strategy for lung adenocarcinoma. Immunohistochemical (IHC) staining with EGFR mutation-specific antibodies of del E746-A750 in exon 19 and L858R in exon 21 has been evaluated in resection specimens in a few studies but rarely in biopsy samples. A total of 169 cases (78 biopsies and 91 resected specimens) of lung adenocarcinoma with EGFR mutation status predefined by direct DNA sequencing were histologically examined, and IHC was performed using EGFR mutation-specific antibodies of del E746-A750 and L858R. The cases with positive results by IHC but negative results by direct DNA sequencing were examined by amplified refractory mutation system. Our results showed that the frequency of EGFR mutations for both E746-A750 deletion and L858R mutation was 38.5% (65/169) by DNA sequencing or amplified refractory mutation system and 34.3% (58/169) by IHC in lung adenocarcinomas. Based on molecular test results, the overall sensitivity, specificity, positive predictive value, and negative predictive value of IHC using these 2 antibodies in all (biopsy/resection) cases were 87.7% (80%/94.3%), 99.0% (97.9%/100%), 98.3% (96%/100%), and 92.8% (88.7%/96.6%), respectively. Lung adenocarcinomas with a predominant acinar, papillary, lepidic, or solid growth pattern more often harbor EGFR mutation of del E746-A750 or L858R. In conclusion, the immunostaining with EGFR del E746-A750 and L858R mutation antibodies is a reliable screening method with high specificity and sensitivity for identifying the EGFR mutation in both resected and biopsied lung adenocarcinomas.

T-cell factor (TCF) and lymphoid enhancer factor (LEF) family transcriptional factors are important nuclear mediators of Wnt/β-catenin signaling. Their roles in cardiac development and diseases remain to be determined. We found that these TCF/LEF family members, including LEF1, TCF7, TCF7L1, and TCF7L2 had unique expression pattern during mouse heart development. At embryonic day (E) 13.5, LEF1 was one of the most abundant TCF/LEF members at mRNA level in the murine heart. By immunohistochemical staining, mesenchymal cells in valvular regions had much stronger intensity of LEF1 than cardiomyocytes in ventricles. Cardiomyocytes with weak to moderate nuclear staining for LEF1 were scattered in compact and trabecular layers. At E17.5, LEF1 mRNA levels dropped dramatically and its protein was no longer detected in cardiomyocytes. TCF7L1 mRNA levels also decreased from E13.5 to E17.5. Weak cytoplasmic, but not nuclear TCF7L1 signal was detected in cardiomyocytes at E13.5 by immunochemistry. TCF7 mRNA was the lowest among all four TCF/LEF members at E13.5 and it further decreased at E17.5. TCF7 protein was only detected by immunohistochemistry in mesothelial and endothelial cells, but not in cardiomyocytes or mesenchymal cells. TCF7L2 mRNA became the most abundant TCF/LEFs at E17.5. Immunolocalization revealed that TCF7L2 formed an intensity gradient with the highest levels at the inner trabecular layer, opposite to the distribution of β-catenin. Wnt signaling activation by cardiac deletion of adenomatosis polyposis coli ( Apc ), a negative Wnt regulator, led to a dramatic increase in cyclin D2, Bmp4, LEF1, and TCF7. BMP mediators, SMAD1/5/8, were increasingly phosphorylated. Chromatin immunoprecipitation revealed that TCF7L2 was removed from and replaced by LEF1 and TCF7 in the promoter of cyclin D2 and Bmp4 to promote β-catenin recruitment. Furthermore, there was co-occupancy of TCF/LEF and SMAD in the adjacent TCF/LEF and SMAD binding sites of Wnt target genes. Finally, we proved that TCF7L2 was a transcriptional suppressor of cyclin D2 and Bmp 4 in a cardiac cell line by overexpression and knockdown experiments. Our results indicate that TCF7L2 is transcriptional repressor of Wnt/β-Catenin pathway in the heart.

Background— How canonical Wnt/β-catenin signals in adult hearts, especially in different diseased states, remains unclear. The proto-oncogene, c-Myc, is a Wnt target and an early response gene during cardiac stress. It is not clear whether c-Myc is activated or how it is regulated during heart failure. Methods and Results— We investigated canonical Wnt/β-catenin signaling and how it regulated c-Myc expression in failing hearts of human ischemic heart disease, idiopathic dilated cardiomyopathy, and murine desmin–related cardiomyopathy. Our data demonstrated that canonical Wnt/β-catenin signaling was activated through nuclear accumulation of β-catenin in idiopathic dilated cardiomyopathy, ischemic heart disease, and murine desmin–related cardiomyopathy when compared with nonfailing controls and transcription factor 7-like 2 (TCF7L2) was the main β-catenin partner of the T-cell factor (TCF) family in adult hearts. We further revealed that c-Myc mRNA and protein levels were significantly elevated in failing hearts by real-time reverse transcription polymerase chain reaction, Western blotting, and immunohistochemical staining. Immunoprecipitation and confocal microscopy further showed that β-catenin interacted and colocalized with TCF7L2. More importantly, chromatin immunoprecipitation confirmed that β-catenin and TCF7L2 were recruited to the regulatory elements of c-Myc. This recruitment was associated with increased histone H3 acetylation and transcriptional upregulation of c-Myc. With lentiviral infection, TCF7L2 overexpression increased c-Myc expression and cardiomyocyte size, whereas shRNA-mediated knockdown of TCF7L2 suppressed c-Myc expression and cardiomyocyte growth in cultured neonatal rat cardiomyocytes. Conclusions— This study indicates that TCF7L2 mediates canonic Wnt/β-catenin signaling and c-Myc upregulation during abnormal cardiac remodeling in heart failure and suppression of Wnt/β-catenin to c-Myc axis can be explored for preventing and treating heart failure.

Glypican-3 (GPC3) is an oncofetal protein that has been demonstrated to be a useful diagnostic immunomarker for hepatocellular carcinoma and hepatoblastoma. Its expression in mesenchymal tumors of the liver, particularly undifferentiated embryonal sarcoma (UES) and mesenchymal hamartoma (MH), has not been investigated. In this study, a total of 24 UESs and 18 MHs were immunohistochemically stained for GPC3 expression. The results showed cytoplasmic staining for GPC3 in 14 (58%) UESs, of which 6 exhibited diffuse immunoreactivity and the remaining 8 showed focal positivity. The patients with GPC3-positive UES tended to be younger (mean 18 years; median 11 years) than those with GPC3-negative tumors (mean 39.4 years; median 27 years), although the difference did not reach statistical significance (P = .06). Eight MHs also exhibited GPC3 immunoreactivity (44%; 4 diffuse and 4 focal). Positive staining in all 8 cases was primarily seen in entrapped nonlesional hepatocytes with a canalicular and cytoplasmic staining pattern. In only 4 cases (22%) was GPC3 immunoreactivity also observed in the mesenchymal component. The patients with positive staining also tended to be younger (mean 2.6 years; median 1.1 years) compared with those with negative staining (mean 16.3 years; median 4.5 years), but the difference was not statistically significant (P = .15). Our data demonstrate that GPC3 is expressed in a subset of UES and MH of the liver. Caution should thus be exercised when evaluating a GPC3-expressing hepatic neoplasm, particularly on a needle biopsy when the differential diagnosis includes poorly differentiated hepatocellular carcinoma or hepatoblastoma.

Global profiling of histone changes in some human cancers demonstrated that loss of histone H4 acetylation at lysine16 (H4KA16) and trimethylation at lysine 20 (H4KM20) was a common hallmark of cancer. It is not clear whether these epigenetic changes also exist in neuroendocrine carcinomas. We semiquantitatively analyzed 32 cases of lung neuroendocrine tumors (LNETs) immunohistochemically stained with H4KA16, H4KM20, and Ki67 antibodies by calculating cumulative scores based on the sum of the product of nuclear stain intensity (1-3) and percentages of positive cells in each category. H4KA16 and H4KM20 levels were compared among typical carcinoid (TC, 11), atypical carcinoid (AC, 6), large cell neuroendocrine carcinoma (LCNEC, 8), and small cell lung cancer (SCLC, 7) and correlated with histologic types and Ki67 labeling. Data were presented as mean±standard error of the mean and statistically analyzed by 1-way analysis of variance and Holm-Sidak method. Normal bronchiolar epithelium had relatively uniform and strong +3 positivity of H4KM20 and H4KA16, which was considered as internal positive controls. This uniformity, however, was gradually lost from low to high grades of LNETs. Semiquantitative analysis revealed that there were significant differences in cumulative scores of H4KA16 (TC, 2.36±0.03; AC, 2.04±0.08; LCNEC, 1.58±0.07; SCLC 1.32±0.05) among LNETs. For H4KM20, significant differences were only observed between low grade (TC, 2.49±0.05 and AC, 2.24±0.09) and high grade (LCNEC, 1.58±0.10 and SCLC 1.68±0.11) LNETs, but not within low or high grade LNETs. The Ki67 cumulative scores (TC, 0.06±0.02; AC, 0.41±0.08; LCNEC, 1.29±0.09; SCLC 1.83±0.06) were inversely correlated with both cumulative H4KA16 and H4KM20 scores by Pearson correlation. We conclude that progressive loss of H4KA16 and H4KM20 from low to high grade LNETs reflects the degree of differentiation and proliferative activity. These histone modifications may serve as tumor biomarkers that may aid in diagnosis and prediction of clinical outcome.

Approximately 5% to 15% of central nervous system metastases by solid tumors spread to the leptomeninges. Transmembrane MUC16 is thought to facilitate anchoring of metastases to other tissues particularly those expressing mesothelin such as pleura and peritoneum. Recently, we have demonstrated that mesothelin is also expressed in the leptomeninges. Mesothelin has a high affinity for MUC16 and mesothelin expressed by some adenocarcinomas. In this study, we evaluated MUC16 and mesothelin immunoreactivity by immunohistochemistry in 11 adenocarcinomas to the leptomeninges, 24 to mesothelin-negative brain, 8 metastases to mesothelin-expressing peritoneum/pleura, 22 to mesothelin-negative peripheral tissues, and 24 with no metastases. MUC16 was detected in 36% of leptomeningeal metastases and 10% of metastases to the brain. Adenocarcinoma metastases to mesothelin-expressing peritoneum or pleura exhibited extensive MUC16 in 75% of cases. In adenocarcinomas with local metastases to lymph nodes or no metastases, 53% or 38% had MUC16 immunoreactivity, respectively. Mesothelin-immunoreactivity was detected in 9% of metastases to the leptomeninges, 17% to the brain, none of the metastases to pleura/peritoneum, 50% of adenocarcinomas with local metastases to lymph nodes, etc, and 33% of adenocarcinomas without metastases. Mesothelin expression was significantly more common in nonmetastatic adenocarcinomas than in metastases to the pleura/peritoneum or leptomeninges. Our findings suggest that adenocarcinomas with MUC16 expression may have an increased risk for metastases to pleura/peritoneum but not the leptomeninges or brain.

Clear cell carcinoma is an uncommon subtype of ovarian carcinoma, accounting for 10% of cases. Clear cell carcinoma typically presents with stage I or II disease, and in this setting prognostic markers could aid in management decisions, in particular the decision to treat with adjuvant chemotherapy. We tested whether expression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3, also known as IMP3) can serve as a new biomarker to predict outcome for patients with clear cell carcinoma and other subtypes of ovarian carcinoma. The expression of IGF2BP3 was evaluated by immunohistochemistry in 475 ovarian carcinomas of different subtypes and correlated with disease-specific survival. IGF2BP3 antibody specificity was validated by correlation of IGF2BP3 protein with mRNA expression level in a series of 35 ovarian carcinomas (r=0.849, P

Merkel cell carcinoma is an uncommon and aggressive primary cutaneous neuroendocrine carcinoma with a high rate of recurrence and metastasis. Optimal management is controversial; consequently, it is imperative to identify the signaling pathways involved in the pathogenesis of Merkel cell carcinoma so that effective therapeutic targeting agents can be developed. We previously reported that K homology domain-containing protein overexpressed in cancer is expressed in high-grade neuroendocrine carcinomas of the lung and extrapulmonary small cell carcinomas. The K homology domain-containing protein overexpressed in cancer (KOC), also known as L523S and IMP-3, is an insulin-like growth factor II messenger RNA-binding protein that promotes tumor cell proliferation by enhancing insulin-like growth factor II protein expression. Expression of KOC in Merkel cell carcinoma has not been investigated. We studied 20 Merkel cell carcinomas by immunohistochemistry using a monoclonal antibody against L523S/KOC. Of 20 Merkel cell carcinomas, 18 (90%) overexpressed KOC, with 11 (55%) overexpressing KOC in greater than 90% of tumor cells, 3 (15%) overexpressing KOC in 50% to 90% of tumor cells, 3 (15%) overexpressing KOC in 10% to 50% of tumor cells, and 1 (5%) overexpressing KOC in less than 10% of tumor cells. The immunostaining intensity was variable, with moderate to strong staining in 14 cases and weak staining in the remaining 4. Extent of expression of K homology domain-containing protein overexpressed in cancer predicted metastasis (P = .04) and was weakly correlated with increased tumor size (P = .08). In conclusion, Merkel cell carcinoma expresses K homology domain-containing protein overexpressed in cancer with an expression pattern similar to high-grade neuroendocrine carcinomas of the lung and extrapulmonary small cell carcinomas. We propose K homology domain-containing protein overexpressed in cancer as a potential target molecule for the treatment of high-grade neuroendocrine carcinomas, irrespective of anatomical location, and a potential marker to predict metastatic disease.

RNA-binding protein IMP3 is a KH-domain-containing protein and a member of the insulin-like growth factor messenger RNA-binding protein family. It is identical to K-homology protein overexpressed in cancer that was identified through screening for genes differentially expressed between benign pancreatic tissue and pancreatic cancer. Several studies have shown that IMP3 is associated with aggressive and advanced tumors in various organs. We studied the expression of IMP3 in benign urothelium and urothelial tumors by immunohistochemistry. The expression pattern of IMP3 was further compared with that of p53 and p16. Our study shows that IMP3 is generally not expressed in benign urothelium or low-grade urothelial tumors including urothelial dysplasia, papillary urothelial neoplasm of low malignant potential, and low-grade papillary urothelial carcinoma. The expression of IMP3 is significantly increased in high-grade urothelial tumors including high-grade papillary urothelial carcinoma, urothelial carcinoma in situ, and invasive urothelial carcinoma. Expression of IMP3 in urothelial tumors parallels the accumulation of nuclear p53, although there is not always a one to one correlation. In contrast, expression of p16 in the different groups of urothelial tumors is more variable. Urothelial carcinomas with invasion of muscularis propria appear to express IMP3 more frequently than lower-stage tumors. These findings suggest that IMP3 may be involved in the progression of urothelial tumors from low grade to high grade in both papillary and flat lesions. Immunohistochemical detection of the combined expression of IMP3 and p53 is useful in the diagnosis of high-grade urothelial tumors, particularly in small, superficial materials.

A number of malignancies, including high-grade neuroendocrine carcinomas of the lung, have been reported to express K homology domain containing protein overexpressed in cancer (KOC), a member of the insulin-like growth factor messenger RNA-binding protein (IMP) family also known as L523S and IMP3. KOC acts to promote tumor cell proliferation by enhancing insulin-like growth factor-II protein expression. This study aimed to examine KOC expression pattern in extrapulmonary neuroendocrine tumors. Seventy-five extrapulmonary neuroendocrine tumors that were surgically resected or had undergone biopsy, including 53 small cell carcinomas (uterine cervix, 21; bladder, 10; colorectum, 13; prostate, 7; stomach, 1; and esophagus, 1) and 22 carcinoid tumors (colorectum, 10; appendix, 5; ileum, 4; duodenum, 2; and stomach, 1), were immunohistochemically studied using a monoclonal antibody against KOC. Our results demonstrated that 47 small cell carcinomas (89%) showed moderate to strong positive staining for KOC, with 25 cases (53%) showing positivity in more than 90% of tumor cells and 22 cases (47%) in 40% to 80% of tumor cells. Three cases showed weak staining in 5% to 10% of the tumor cells. The remaining 3 cases (uterine cervix, 2; bladder, 1) showed completely negative immunoreactivity. No KOC immunostaining was detected in 22 carcinoid tumors. These findings indicate that KOC may play an important role in the regulation of biologic behavior of extrapulmonary small cell carcinomas. In addition, immunohistochemical detection of KOC expression may serve as a useful diagnostic tool in the distinction between small cell carcinoma and carcinoid tumor, particularly when the diagnostic material is a small biopsy with crushing artifact.

Substantial evidence suggests that the progressive loss of cardiomyocytes caused by apoptosis significantly contributes to the development of heart failure. beta-Adrenergic receptor activation and subsequent persistent phosphodiesterase 3A (PDE3A) downregulation and concomitant inducible cAMP early repressor (ICER) upregulation (PDE3A/ICER feedback loop) has been proposed to play a key role in the pathogenesis of cardiomyocyte apoptosis. In contrast, insulin-like growth factor-1 can activate cell survival pathways, providing protection against cell death and restoring muscle function. In this study, we found that insulin-like growth factor-1 activates extracellular signal-regulated kinase 5 (ERK5) and inhibits PDE3A/ICER feedback loop. Insulin-like growth factor-1 normalized isoproterenol-mediated PDE3A downregulation and ICER upregulation via ERK5/MEF2 activation, and also inhibited isoproterenol-induced myocyte apoptosis. To determine the physiological relevance of ERK5 activation in regulating PDE3A/ICER feedback loop, we investigated the PDE3A/ICER expression and cardiomyocyte apoptosis in transgenic mice with cardiac specific expression of a constitutively active form of mitogen-activated protein (MAP)/extracellular signal-regulated protein kinase (ERK) kinase 5alpha (MEK5alpha) (CA-MEK5alpha-Tg). In wild-type mice, pressure overload- or doxorubicin-induced significant reduction of PDE3A expression and subsequent ICER induction. Cardiac specific expression of CA-MEK5alpha rescued pressure overload- or doxorubicin-mediated PDE3A downregulation and ICER upregulation and inhibited myocyte apoptosis as well as subsequent cardiac dysfunction in vivo. These data suggest that preventing the feedback loop of PDE3A/ICER by ERK5 activation could inhibit progression of myocyte apoptosis as well as cardiac dysfunction. These data suggest a new therapeutic paradigm for end stage of heart failure by inhibiting the PDE3A/ICER feedback loop via activating ERK5.

Malakoplakia is a rare inflammatory disorder that results from an infectious process and that may be associated with immunosuppression. It most often occurs in the genitourinary tract; however, isolated cases have been reported in many other organs, including colon, stomach, lung, liver, bone, uterus, and skin. In the bladder, which is the most common site of involvement, it may be mistaken for malignancy on cystoscopic examination or computerized topography scan. Gram-negative bacteria such as Escherichia coli and Klebsiella pneumonia are often isolated from malakoplakia lesions, although other less common bacteria such as Rhodococcus equi may also be found. This is a case report of prostatic malakoplakia diagnosed on needle core biopsy from a patient suspected to harbor a malignancy.

Hepatoid carcinomas are tumors that display, at least focally, cytologic and/or architectural features of hepatocellular carcinoma. They have been described in several organs, most notably in the stomach and ovary. We report a case of hepatoid carcinoma of the pancreas that developed in a 41-year-old woman in association with a pancreatic endocrine carcinoma. The fine needle aspiration material was characterized by the presence of monotonous, small-to-medium sized tumor cells with round nuclei and finely granular chromatin, intermixed with more atypical tumor cells displaying larger nuclei with coarse clumped chromatin, prominent nucleoli, and moderate amounts of foamy cytoplasm. The excised specimen displayed a poorly differentiated pancreatic endocrine carcinoma associated with well-defined islands of larger tumor cells growing in a perisinusoidal pattern which, based on their immunohistochemical profile and the demonstration of bile, proved to represent a hepatoid component. This case and prior examples in the literature suggest that hepatoid carcinomas of the pancreas appear to be a heterogeneous group of tumors (pure or associated with another histologic component) that are often associated with early liver metastasis and a short survival, although those arising as a component of endocrine tumors seem to fare slightly better. Hepatoid carcinoma of the pancreas should be included in the differential diagnosis of pancreatic tumors composed of large eosinophilic cells.

A key feature of lung fibrosis is the accumulation of myofibroblasts. Interleukin 13 (IL-13) is a pro-fibrotic mediator that directly and indirectly influences the activation of myofibroblasts. Transforming growth factor beta (TGF-β) promotes the differentiation of fibroblasts into myofibroblasts, and can be regulated by IL-13. However, IL-13’s downstream signaling pathways are not completely understood. We previously reported that the transcription factor Yin Yang 1 (YY1) is upregulated in fibroblasts treated with TGF-β and in the lungs of mice and patients with pulmonary fibrosis. Moreover, YY1 directly regulates collagen and alpha smooth muscle actin (α-SMA) expression in fibroblasts. However, it is not known if IL-13 regulates fibroblast activation through YY1 expression. We hypothesize that IL-13 up-regulates YY1 expression through regulation of AKT activation, leading to fibroblast activation. In this study we found that YY1 was upregulated by IL-13 in lung fibroblasts in a dose- and time-dependent manner, resulting in increased α-SMA. Conversely, knockdown of YY1 blocked IL-13-induced α-SMA expression in fibroblasts. Furthermore, AKT phosphorylation was increased in fibroblasts treated with IL-13, and AKT overexpression upregulated YY1, whereas blockade of AKT phosphorylation suppressed the induction of YY1 by IL-13 in vitro. In vivo YY1 was upregulated in fibrotic lungs from CC10-IL-13 transgenic mice compared to that from wild-type littermates, which was associated with increased AKT phosphorylation. Taken together, these findings demonstrate that IL-13 is a potent stimulator and activator of fibroblasts, at least in part, through AKT-mediated YY1 activation.

Immune-mediated lung injury is an important component of Pneumocystis pneumonia (PcP)-related immunorestitution disease (IRD). However, the individual contribution of CD4+and CD8+T cells to the pathophysiology of IRD remains undetermined. Therefore, IRD was modeled in severe combined immunodeficient mice, and specific T cell depletion was used to determine how T cell subsets interact to affect the nature and severity of disease. CD4+cells were more abundant than CD8+cells during the acute stage of IRD that coincided with impaired pulmonary physiology and organism clearance. Conversely, CD8+cells were more abundant during the resolution phase following P. carinii clearance. Depletion of CD4+T cells protected mice from the acute pathophysiology of IRD. However, these mice could not clear the infection and developed severe PcP at later time points when a pathological CD8+T cell response was observed. In contrast, mice depleted of CD8+T cells efficiently cleared the infection but developed more severe disease, an increased frequency of IFN-γ-producing CD4+cells, and a prolonged CD4+T cell response than mice with both CD4+and CD8+cells. These data suggest that CD4+T cells mediate the acute respiratory disease associated with IRD. In contrast, CD8+T cells contributed to neither lung injury nor organism clearance when CD4+cells were present, but instead served to modulate CD4 function. In the absence of CD4+cells, CD8+T cells produced a nonprotective, pathological immune response. These data suggest that the interplay of CD4+and CD8+T cells affects the ultimate outcome of PcP-related IRD.

Our in vitro studies revealed that a transcription factor, Forkhead box protein O1 (FoxO1), negatively regulates the expression of NaV1.5, a main α subunit of the cardiac Na(+) channel, by altering the promoter activity of SCN5a in HL-1 cardiomyocytes. The in vivo role of FoxO1 in the regulation of cardiac NaV1.5 expression remains unknown. The present study aimed to define the role of FoxO1 in the regulation of NaV1.5 expression and cardiac Na(+) channel activity in mouse ventricular cardiomyocytes and assess the cardiac electrophysiological phenotype of mice with cardiac FoxO1 deletion. Tamoxifen-induced and cardiac-specific FoxO1 deletion was confirmed by polymerase chain reaction (PCR). Cardiac FoxO1 deletion failed to result in either cardiac functional changes or hypertrophy as assessed by echocardiography and individual ventricular cell capacitances, respectively. Western blotting showed that FoxO1 was significantly decreased while NaV1.5 protein level was significantly increased in mouse hearts with FoxO1 deletion. Reverse transcription-PCR (RT-PCR) revealed that FoxO1 deletion led to an increase in NaV1.5 and Na(+) channel subunit β3 mRNA, but not β1, 2, and 4, or connexin 43. Whole patch-clamp recordings demonstrated that cardiac Na(+) currents were significantly augmented by FoxO1 deletion without affecting the steady-state activation and inactivation, leading to accelerated depolarization of action potentials in mouse ventricular cardiomyocytes. Electrocardiogram recordings showed that the QRS complex was significantly shortened and the P wave amplitude was significantly increased in conscious and unrestrained mice with cardiac FoxO1 deletion. NaV1.5 expression was decreased in the peri-infarct (border-zone) of mice with myocardial infarction and FoxO1 accumulated in the cardiomyocyte nuclei of chronic ischemic human hearts. Our findings indicate that FoxO1 plays an important role in the regulation of NaV1.5 and β3 subunit expressions as well as Na(+) channel activity in the heart and that FoxO1 is involved in the modulation of NaV1.5 expression in ischemic heart disease.

In the experiments here, the time- and voltage-dependent properties of the Ca2+-independent, depolarization-activated K+ currents in adult mouse ventricular myocytes were characterized in detail. In the majority (65 of 72, approximately 90%) of cells dispersed from the ventricles, analysis of the decay phases of the outward currents revealed three distinct K+ current components: a rapidly inactivating, transient outward K+ current, Ito,f (mean +/- SEM taudecay = 85 +/- 2 ms); a slowly (mean +/- SEM taudecay = 1,162 +/- 29 ms) inactivating K+ current, IK,slow; and a non inactivating, steady state current, Iss. In a small subset (7 of 72, approximately 10%) of cells, Ito,f was absent and a slowly inactivating (mean +/- SEM taudecay = 196 +/- 7 ms) transient outward current, referred to as Ito,s, was identified; the densities and properties of IK,slow and Iss in Ito,s-expressing cells are indistinguishable from the corresponding currents in cells with Ito,f. Microdissection techniques were used to remove tissue pieces from the left ventricular apex and from the ventricular septum to allow the hypothesis that there are regional differences in Ito,f and Ito,s expression to be tested directly. Electrophysiological recordings revealed that all cells isolated from the apex express Ito,f (n = 35); Ito,s is not detected in these cells (n = 35). In the septum, by contrast, all of the cells express Ito,s (n = 28) and in the majority (22 of 28, 80%) of cells, Ito,f is also present. The density of Ito,f (mean +/- SEM at +40 mV = 6.8 +/- 0.5 pA/pF, n = 22) in septum cells, however, is significantly (P0.001) lower than Ito,f density in cells from the apex (mean +/- SEM at +40 mV = 34.6 +/- 2.6 pA/pF, n = 35). In addition to differences in inactivation kinetics, Ito,f, Ito,s, and IK,slow display distinct rates of recovery (from inactivation), as well as differential sensitivities to 4-aminopyridine (4-AP), tetraethylammonium (TEA), and Heteropoda toxin-3. IK,slow, for example, is blocked selectively by low (10-50 microM) concentrations of 4-AP and by (/=25 mM) TEA. Although both Ito,f and Ito,s are blocked by high (100 microM) 4-AP concentrations and are relatively insensitive to TEA, Ito,f is selectively blocked by nanomolar concentrations of Heteropoda toxin-3, and Ito,s (as well as IK,slow and Iss) is unaffected. Iss is partially blocked by high concentrations of 4-AP or TEA. The functional implications of the distinct properties and expression patterns of Ito,f and Ito,s, as well as the likely molecular correlates of these (and the IK,slow and Iss) currents, are discussed.

Lu Xiao, Haiqing Bai, James Boyer, Bo Ye, Ning Hou, Haodong Xu, and Faqian Li Department of Pathology and Laboratory Medicine and Cardiovascular Research Institute, University of Rochester Medical Center, Rochester, NY, USA Backgrounds: Canonical Wnt signaling appears to have multiphasic and often antagonistic roles in cardiac development. The molecular mechanism for these opposing actions is not clear. We hypothesized that alternative splicing of TCF7L2, a nuclear interaction partner of beta-catenin is involved in the specificity of canonical Wnt signaling. Methods: RT-PCR were performed on embryonic (E16.5) and neonatal (day 8) hearts with primers spanning the end of first exon and the beginning of last exon and the products were cloned and sequenced. Result: There are totally 18 exons identified so far in TCF7L2. We sequenced 56 clones and 53 clones (29 from day 8) and (24 from E16.5) contained TCF7L2 sequences. No exon 6 or exon 17 was found in TCF7L2 transcripts of mouse hearts. Most clones (more than 80%) from E16.5 and day 8 hearts excluded exon 4. Both E16.5 and day 8 hearts had one clone with exon 9 deletion which does not change reading frame and another with alterations in exon 3 that lead to reading frame shift and premature stop codon. As reported in other organs, there were extensive alternative splicing in the C-terminal exons 14, 15 and 16. The inclusion of exon 14 was more frequently in day 8 (18 of 29, 62%) than in E16.5 (8 of 24, 33%) hearts. The peptide encoded by exon 14 has conserved functional motif. Additionally, this alternative exon usage can change the C-terminus of TCF7L2 to include or exclude the so-called E tail with two binding motifs for C-terminal binding protein. Conclusion: The isoform switch of TCF7L2 occurs in neonatal mouse hearts and may have a role in the terminal differentiation of cardiac myocytes during this period.

Backgrounds: DNA damage occurs in cardiomyocytes during normal cellular metabolism and is significantly increased under cardiac stresses. How cardiomyocytes repair their DNA damage, especially DNA double strand breaks (DSBs), remains undetermined. We assessed DSBs caused by oxidative stress. More importantly, we investigated the spatiotemporal dynamics of DNA repair protein assembly/disassembly in DNA damage sites. Methods: Cultured neonatal rat cardiomyocytes were treated with different doses of hydrogen peroxide (H2O2) for 30 minutes to assess DNA damage response (DDR). To investigate the dynamics of DDR, cells were treated with 200 uM H2O2 and followed up to 72 hours. DSBs were evaluated by counting DNA damage foci after staining with antibody against histone H2AX phosphorylation at serine 139 (g-H2AX). The dynamics and posttranslational modification of DNA repair proteins were determined by Western blotting, immunolabeling, and confocal microscopy. Result: g-H2AX was proportionally increased to H2O2 dosage. Discrete nuclear g-H2AX foci were seen 30 minutes after hydrogen peroxide treatment with 50 uM, but became pannuclear when H2O2 was above 400 uM. At 200 uM of hydrogen peroxide, g-H2AX started to increase at 15 minutes and reached to highest levels at 60 minutes with up to 70 nuclear foci, started to decline at 2 hours, and returned to basal levels at 24 hours. DDR transducer kinase, ataxia telangiectasia mutated (ATM) was activated at 5 minutes with increased phosphorylation at serine 1981 (pATM) which started to decrease at 24 hours, but remained elevated up to 48 hours. Another DDR transducer kinase, ATM and Rad3-related (ATR) showed a biphasic activation at 30 minutes and 8 hours. ATM and ATR colocalized with g-H2AX. DNA damage mediator proteins such as MRN complex and p53BP1 were also recruited to sites of DNA damage at g-H2AX foci. Conclusions: DSBs and their repair have emerged as a new frontier of stress responses. Newly developed methods for studying g-H2AX and DNA repair protein dynamics can be explored to investigate DDR to oxidative stress in cardiomyocytes.

In the experiments here, the developmental expression of the functional Ca(2+)-independent, depolarization-activated K+ channel currents, Ito and IK, and of the voltage-gated K+ channel (Kv) alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2 in rat ventricular myocytes were examined quantitatively. Using the whole-cell patch clamp recording method, the properties and the densities of Ito and IK in ventricular myocytes isolated from postnatal day 5 (P5), 10 (P10), 15 (P15), 20 (P20), 25 (P25), 30 (P30), and adult (8-12 wk) rats were characterized and compared. These experiments revealed that mean Ito densities increase fourfold between birth and P30, whereas IK densities vary only slightly. Neither the time- nor the voltage-dependent properties of the currents vary measurably, suggesting that the subunits underlying functional Ito and IK channels are the same throughout postnatal development. In parallel experiments, the developmental expression of each of the voltage-gated K+ channel alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2, was examined quantitatively at the mRNA and protein levels using subunit-specific probes. RNase protection assays revealed that Kv1.4 message levels are high at birth, increase between P0 and P10, and subsequently decrease to very low levels in adult rat ventricles. The decrease in message is accompanied by a marked reduction in Kv1.4 protein, consistent with our previous suggestion that Kv1.4 does not contribute to the formation of functional K+ channels in adult rat ventricular myocytes. In contrast to Kv1.4, the mRNA levels of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 increase (three- to five-fold) between birth and adult. Western analyses, however, revealed that the expression patterns of these subunits proteins vary in distinct ways: Kv1.2 and Kv4.2, for example, increase between P5 and adult, whereas Kv1.5 remains constant and Kv2.1 decreases. Throughout development, therefore, there is a mismatch between the numbers of Kv alpha subunits expressed and the functional voltage-gated K+ channel currents distinguished electrophysiologically in rat ventricular myocytes. Alternative experimental approaches will be required to define directly the Kv alpha subunits that underlie functional voltage-gated K+ channels in these (and other) cells. In addition, the finding that Kv alpha subunit protein expression levels do not necessarily mirror mRNA levels suggests that caution should be exercised in attempting functional interpretations of observed changes in mRNA levels alone.

Lung injury, whether induced by infection or caustic chemicals, initiates a series of complex wound-healing responses. If uncontrolled, these responses may lead to fibrotic lung diseases and loss of function. Thus, resolution of lung injury must be tightly regulated. The key regulatory proteins required for tightly controlling the resolution of lung injury have yet to be identified. Here we show that loss of deubiquitinase CYLD led to the development of lung fibrosis in mice after infection with Streptococcus pneumoniae. CYLD inhibited transforming growth factor-β-signalling and prevented lung fibrosis by decreasing the stability of Smad3 in an E3 ligase carboxy terminus of Hsc70-interacting protein-dependent manner. Moreover, CYLD decreases Smad3 stability by deubiquitinating K63-polyubiquitinated Akt. Together, our results unveil a role for CYLD in tightly regulating the resolution of lung injury and preventing fibrosis by deubiquitinating Akt. These studies may help develop new therapeutic strategies for preventing lung fibrosis., Lung injury initiates a series of wound-healing responses, which if unregulated, can lead to fibrosis. Li et al. show that the deubquitinase CYLD has a key role in the prevention of fibrosis by inhibiting transforming growth factor β-signalling through the direct deubiquitination of the protein kinase Akt.

Background Failed pediatric heart allografts with diastolic dysfunction exhibit severe epicardial fibrosis. The molecular mechanism underlying this process is poorly understood. Canonical Wnt/β-catenin signaling plays an important role in epithelial–mesenchymal transition and is implicated in fibrosing diseases. In this study, we tested the hypothesis that canonical Wnt/β-catenin signaling is activated in epicardial fibrosis of end-stage dysfunctional pediatric allografts. Methods Fourteen explanted heart grafts of 12 patients who had undergone 14 heart transplantations were used for immunohistochemical staining of β-catenin and its nuclear binding partners, T-cell factor/lymphoid enhancer factor family transcriptional factors. Fourteen age-matched native hearts from patients who had undergone first heart transplantation without evidence of epicardial fibrosis were used as controls. Results and conclusions Epicardial fibroblasts from explanted allografts demonstrated nuclear accumulation of β-catenin. These cells also showed nuclear positivity for T-cell factor 4. No T-cell factor 3 expression was present in the epicardium. T-cell factor 1 and lymphoid enhancer factor 1 were observed in lymphocytes, but not in other cell types of the epicardium. These findings suggest an association between canonical Wnt/beta-catenin signaling and epicardial fibrosis of failed pediatric heart allografts. Should activation of this pathway be shown to be causal to epicardial fibrosis in this setting, then inhibition of this pathway may help to prevent this devastating process.

Na(V)1.5 is a cardiac voltage-gated Na(+) channel αsubunit and is encoded by the SCN5a gene. The activity of this channel determines cardiac depolarization and electrical conduction. Channel defects, including mutations and decrease of channel protein levels, have been linked to the development of cardiac arrhythmias. The molecular mechanisms underlying the regulation of Na(V)1.5 expression are largely unknown. Forkhead box O (Foxo) proteins are transcriptional factors that bind the consensus DNA sequences in their target gene promoters and regulate the expression of these genes. Comparative analysis revealed conserved DNA sequences, 5'-CAAAACA-3' (insulin responsive element, IRE), in rat, mouse and human SCN5a promoters with the latter two containing two overlapping Foxo protein binding IREs, 5'-CAAAACAAAACA-3'. This finding led us to hypothesize that Foxo1 regulates Na(V)1.5 expression by directly binding the SCN5a promoter and affecting its transcriptional activity. In the present study, we determined whether Foxo1 regulates Na(V)1.5 expression at the transcriptional level and also defined the role of Foxo1 in hydrogen peroxide (H(2)O(2))-mediated Na(V)1.5 suppression in HL-1 cardiomyocytes using chromatin immunoprecipitation (ChIP), constitutively nuclear Foxo1 expression, and RNAi Foxo1 knockdown as well as whole cell voltage-clamp recordings. ChIP with anti-Foxo1 antibody and follow-up semi-quantitative PCR with primers flanking Foxo1 binding sites in the proximal SCN5a promoter region clearly demonstrated enrichment of DNA, confirming Foxo1 recruitment to this consensus sequence. Foxo1 mutant (T24A/S319A-GFP, Foxo1-AA-GFP) was retained in nuclei, leading to a decrease of Na(V)1.5 expression and Na(+) current, while silencing of Foxo1 expression by RNAi resulted in the augmentation of Na(V)1.5 expression. H(2)O(2) significantly reduced Na(V)1.5 expression by promoting Foxo1 nuclear localization and this reduction was prevented by RNAi silencing Foxo1 expression. These studies indicate that Foxo1 negatively regulates Na(V)1.5 expression in cardiomyocytes and reactive oxygen species suppress Na(V)1.5 expression through Foxo1.

Insulin-like growth factor II-messenger RNA-binding protein 3 (IMP3) is an oncofetal RNA-binding protein that promotes tumor cell proliferation by enhancing IGF-II protein synthesis and inducing cell adhesion and invasion by stabilizing CD44 mRNA. IMP3 expression has been studied in many human neoplasms with growing evidence that IMP3 is a biomarker of enhanced tumor aggressiveness. IMP3 expression has been correlated with a poorer phenotypic profile including increased risk of metastases and decreased survival. Only a few studies have examined IMP3 expression in lung cancers. We review here the literature concerning IMP3 expression in lung neoplasms, specifically adenocarcinoma, squamous cell carcinoma, and neuroendocrine tumors of the lung. IMP3 immunohistochemical expression was reported in 27-55% of cases of primary pulmonary adenocarcinoma and in 75-90% of cases of squamous cell carcinoma of the lung. In adenocarcinoma, IMP3 expression was reported to be correlated with more poorly differentiated histological grade, advanced stage of disease and lymph node metastases. IMP3 expression also may be a marker of high grade pre-invasive squamous lesions including high grade dysplasia and carcinoma in situ. In neuroendocrine tumors of the lung, IMP3 expression was expressed in all reported cases of large cell neuroendocrine carcinoma and small cell lung carcinoma, but expression was limited in carcinoid tumors. Overall, IMP3 appears to be a useful diagnostic marker for lung cancer pathology including for discriminating high grade neuroendocrine tumors and low grade carcinoids and for identifying high grade pre-invasive squamous lesions.

Rationale: The differentiation of fibroblasts into myofibroblasts is a cardinal feature of idiopathic pulmonary fibrosis (IPF). The transcription factor Yin Yang 1 (YY1) plays a role in the proliferation and differentiation of diverse cell types, but its role in fibrotic lung diseases is not known. Objectives: To elucidate the mechanism by which YY1 regulates fibroblast differentiation and lung fibrosis. Methods: Lung fibroblasts were cultured with transforming growth factor (TGF)-β or tumor necrosis factor-α. Nuclear factor (NF)-κB, YY1, and α-smooth muscle actin (SMA) were determined in protein, mRNA, and promoter reporter level. Lung fibroblasts and lung fibrosis were assessed in a partial YY1-deficient mouse and a YY1f/f conditional knockout mouse after being exposed to silica or bleomycin. Measurements and Main Results: TGF-β and tumor necrosis factor-α up-regulated YY1 expression in lung fibroblasts. TGF-β–induced YY1 expression was dramatically decreased by an inhibitor of NF-κB, which blocked I-κB degradation. YY1 is significantly overexpressed in both human IPF and murine models of lung fibrosis, including in the aggregated pulmonary fibroblasts of fibrotic foci. Furthermore, the mechanism of fibrogenesis is that YY1 can up-regulate α-SMA expression in pulmonary fibroblasts. YY1-deficient (YY1+/−) mice were significantly protected from lung fibrosis, which was associated with attenuated α-SMA and collagen expression. Finally, decreasing YY1 expression through instilled adenovirus-cre in floxed-YY1f/f mice reduced lung fibrosis. Conclusions: YY1 is overexpressed in fibroblasts in both human IPF and murine models in a NF-κB–dependent manner, and YY1 regulates fibrogenesis at least in part by increasing α-SMA and collagen expression. Decreasing YY1 expression may provide a new therapeutic strategy for pulmonary fibrosis.

The presence of individual neuroendocrine cells in rare peripancreatic lymph nodes (LNs) suggests that neuroendocrine tumor or nested neuroendocrine cell proliferation can arise in situ from neuroendocrine cells native to any LN. However, it is very difficult to ascertain whether any neuroendocrine lesion in LNs is a primary tumor or a metastasis from adjacent organs. We encountered 4 cases of neuroendocrine proliferation in intrathoracic LNs (ILNs) of patients with primary lung adenocarcinoma. All patients had a single lung mass without mediastinal lymphadenopathy based on computed tomography and positron emission tomography imaging. Mediastinal staging was done by either mediastinoscopy or thoracotomy and none of them had metastasis from adenocarcinoma in any LN. One patient had three ILNs positive for neuroendocrine proliferation measuring 1.7, 1.8, and 4.0 mm, respectively and a minute tumorlet less than 1.0 mm in the lung. Three other patients had small areas of neuroendocrine proliferation no more than 1 mm in single ILN without any lung neuroendocrine lesion. Neuroendocrine cells in ILNs often formed nests of varying size with similar morphology to carcinoid tumorlet in the lung. Small clusters of neuroendocrine cells without any particular pattern were often seen together with these nests. These cells were positive for neuroendocrine markers: synaptophysin, chromogranin, and CD56. They were also positive for CK7 and TTF-1. It is interesting to note, single cells positive for neuroendocrine markers and TTF-1 were identified near or away from these neuroendocrine nests or clusters. These findings suggest that neuroendocrine lesion can be incidentally identified in ILNs. Close clinical follow-up is warranted as metastasis from or synchronous lesions in adjacent organs cannot be excluded.

Enhancer of zeste homolog 2, the catalytic subunit of polycomb repressive complex 2, is a histone methyltransferase and plays an important role in cell proliferation and cell cycle regulation. It has been shown to be overexpressed in a number of malignant neoplasms. This study aimed to determine the expression pattern of enhancer of zeste homolog 2 in neuroendocrine tumors of the lung and the potential of enhancer of zeste homolog 2 to serve as a biomarker to segregate carcinoids from high-grade neuroendocrine carcinomas. Fifty-four cases, including 25 typical carcinoids, 7 atypical carcinoids, 9 large-cell neuroendocrine carcinomas, and 13 small-cell lung carcinomas, were immunohistochemically studied using a monoclonal antibody against enhancer of zeste homolog 2. All 13 small-cell lung carcinomas demonstrated moderate to strong nuclear staining with 12 exhibiting more than 90% of tumor cells staining. All 9 large-cell neuroendocrine carcinomas were moderately to strongly positive for enhancer of zeste homolog 2, with 6 cases having staining in more than 80% of tumor cells. In contrast, all 25 typical carcinoids and 6 atypical carcinoids showed only rare scattered enhancer of zeste homolog 2-positive tumor cells, with 1 case of atypical carcinoid exhibiting moderate staining in 40% of tumor cells. A subsequent validation study of the 14 specimens of lung or mediastinal lymph node biopsy and fine-needle aspiration, including 6 small-cell lung carcinomas, 2 large-cell neuroendocrine carcinomas, 5 typical carcinoids, and 1 atypical carcinoid, was performed. Enhancer of zeste homolog 2 was diffusely and strongly positive in all small-cell lung carcinomas and large-cell neuroendocrine carcinomas, even with severe crush artifact, whereas it was only positive in rare tumor cells in carcinoids. These findings support the formulation that enhancer of zeste homolog 2 may play an important role in the regulation of biologic behavior of high-grade neuroendocrine carcinomas and as a diagnostically useful marker in distinguishing high-grade neuroendocrine carcinomas from carcinoids.

Histopathologic distinction between benign and malignant bile duct epithelial lesions on endoscopic biopsies can be extremely challenging because of limited material, crush artifact, and compounding inflammatory and/or reactive changes particularly after stent placement. In this study, a total of 72 endoscopic bile duct biopsies, including 40 adenocarcinomas and 32 benign cases, were immunohistochemically examined for the expression of S100P, von Hippel-Lindau gene product (pVHL), and IMP3 to evaluate their diagnostic value. The results showed that 36 adenocarcinomas (90%) exhibited strong nuclear and cytoplasmic staining for S100P, of which 30 (83.3%) showed diffuse immunoreactivity. Intermediate to strong cytoplasmic staining for IMP3 was demonstrated in 31 tumors (77.5%) (15 diffuse, 16 focal). Completely negative staining for pVHL was observed in 37 adenocarcinomas. In the remaining 3 tumors, focal (1) or diffuse (2) membranous and cytoplasmic pVHL immunoreactivity was detected. Twenty-eight tumors (70%) showed a S100P+/IMP3+/pVHL- staining pattern, 6 (15%) with a S100P+/IMP3-/pVHL- pattern, and 2 (5%) with a S100P-/IMP3+/pVHL- pattern. All 32 benign biopsies were completely negative for IMP3 with the exception of 2 cases with focal dysplasia where focal immunoreactivity was observed. Thirty benign biopsies (93.8%) were positive for pVHL with a diffuse staining pattern observed in 28 cases (93.3%). Eight benign biopsies (25%) showed focal S100P positivity. Twenty-two benign biopsies (68.8%) displayed a S100P-/IMP3-/pVHL+ staining pattern. In conclusion, an immunohistochemical panel consisting of S100P, pVHL, and IMP3 can be helpful in distinguishing adenocarcinoma from reactive epithelial changes on challenging bile duct biopsies. The findings of focal S100P and/or IMP3 expression with reciprocal loss of pVHL immunoreactivity in a few benign biopsies suggest a use of these markers in the detection of early epithelial dysplasia that may be beyond histologic recognition.

Rationale: Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca 2+ /calmodulin (CaM)-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca 2+ /CaM and cGMP signaling; however, its function in cardiomyocytes is unknown. Objective: Herein, we investigate the role of Ca 2+ /CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo. Methods and Results: Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Furthermore, PDE1A plays a critical role in phenylephrine-induced reduction of intracellular cGMP- and cGMP-dependent protein kinase (PKG) activity and thereby cardiomyocyte hypertrophy in vitro. Conclusions: These results elucidate a novel role for Ca 2+ /CaM-stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG-dependent mechanism, thereby demonstrating Ca 2+ and cGMP signaling cross-talk during cardiac hypertrophy.

Insulin-like growth factor II mRNA binding protein 3 is an oncofetal protein that is expressed in multiple malignancies. This study aimed to determine the correlation of insulin-like growth factor II mRNA binding protein 3 expression with histologic grade of lung adenocarcinoma. Eighty-nine cases, including 11 atypical adenomatous hyperplasias, 10 pure bronchioloalveolar carcinomas, 36 well-differentiated adenocarcinomas and 41 moderately or poorly differentiated adenocarcinomas, were immunohistochemically studied using a monoclonal antibody against insulin-like growth factor II mRNA binding protein 3. Twenty-nine (70.7%) of 41 moderately to poorly differentiated adenocarcinomas were positive for insulin-like growth factor II mRNA binding protein 3, with 26 (89.7%) tumors demonstrating either a strong staining or staining in greater than 30% of tumor cells. Four (40.0%) of 10 bronchioloalveolar carcinomas and 13 (36.1%) of 36 well-differentiated adenocarcinomas exhibited insulin-like growth factor II mRNA binding protein 3 positivity with a variable degree and percentage of tumor cells staining. When bronchioloalveolar carcinomas were present in a pure form or as a component of adenocarcinomas, positive insulin-like growth factor II mRNA binding protein 3 staining was always patchy, with less than 20% of tumor cells stained. Overall, the frequency of positive insulin-like growth factor II mRNA binding protein 3 staining was lower in bronchioloalveolar carcinomas and well-differentiated adenocarcinomas compared to moderately/poorly differentiated adenocarcinomas (P < .01). No insulin-like growth factor II mRNA binding protein 3 signal was detected in any case of atypical adenomatous hyperplasia. These findings show that insulin-like growth factor II mRNA binding protein 3 is strongly and diffusely expressed in a large proportion of moderately/poorly differentiated lung adenocarcinomas, in particular in the solid component of mixed subtype adenocarcinomas, less frequently expressed in well-differentiated adenocarcinomas and bronchioloalveolar carcinomas, and negative in atypical adenomatous hyperplasias. The higher frequency of expression in moderately/poorly differentiated adenocarcinomas suggests that insulin-like growth factor II mRNA binding protein 3 expression may be associated with an aggressive biological behavior.