SLO2.1/NALCN Functional Complex Activity in Mouse Myometrial Smooth Muscle Cells During Pregnancy.

At the end of pregnancy, the uterus transitions from a quiescent to a highly contractile state. This is partly due to depolarization of the resting membrane potential in uterine (myometrial) smooth muscle cells (MSMCs). Experiments with human MSMCs showed that the membrane potential is regulated by a functional complex between the sodium (Na+)-activated potassium (K+) channel SLO2.1 and the Na+ Leak Channel Non-Selective (NALCN). In human MSMCs, Na+ entering through NALCN activates SLO2.1, leading to K+ efflux, membrane hyperpolarization (cells become more negative inside), and reduced contractility. Decreased SLO2.1/NALCN activity results in reduced K+ efflux, leading to membrane depolarization, Ca2+ influx via voltage-dependent calcium channels, and increased MSMC contractility. However, all of these experiments were performed with MSMCs isolated from women at term, so the role of the SLO2.1/NALCN complex early in pregnancy was speculative. To address this question here, we examined the role of the SLO2.1/NALCN complex in regulating mouse MSMC membrane potential across pregnancy. We report that Slo2.1 and Nalcn expression change along pregnancy, being more highly expressed in MSMCs from non-pregnant and early pregnant mice than in those from late-pregnant mice. Functional studies revealed that SLO2.1 channels mediate a significant portion of the K+ current in mouse MSMCs, particularly in cells from non-pregnant and early pregnant mice. Activation of SLO2.1 by Na+ influx through NALCN led to membrane hyperpolarization in MSMCs from early pregnancy but not in MSMCs from later pregnancy. Moreover, we found that the NALCN/SLO2.1 complex regulates intracellular Ca2+ responses more in MSMCs from non-pregnant and early pregnancy mice than in MSMCs from late pregnancy. Together, these findings reveal that the SLO2.1/NALCN functional complex is conserved between mouse and humans and functions throughout pregnancy. This work could open avenues for targeted pharmacological interventions in pregnancy-related complications.

BKCa channels are involved in spontaneous and lipopolysaccharide-stimulated uterine contraction in late gestation mice†.

The large-conductance, voltage-gated, calcium (Ca2+)-activated potassium channel (BKCa) is one of the most abundant potassium channels in the myometrium. Previous work conducted by our group has identified a link between inflammation, BKCa channels and excitability of myometrial smooth muscle cells. Here, we investigate the role of BKCa channels in spontaneous and lipopolysaccharide (LPS)-stimulated uterine contraction to gain a better understanding of the relationship between the BKCa channel and uterine contraction in basal and inflammatory states. Uteri of C57BL/6 J mice on gestational day 18.5 (GD18.5) were obtained and either fixed in formalin or used immediately for tension recording or isolation of primary myocytes for patch-clamp. Paraffin sections were used for immunofluorescenctdetection of BKCa and Toll-like receptor (TLR4). For tension recordings, LPS was administered to determine its effect on uterine contractions. Paxilline, a BKCa inhibitor, was used to dissect the role of BKCa in uterine contraction in basal and inflammatory states. Finally, patch-clamp recordings were performed to investigate the relationship between LPS, the BKCa channel and membrane currents in mouse myometrial smooth muscle cells (mMSMCs). We confirmed the expression of BKCa and TLR4 in the myometrium of GD18.5 mice and found that inhibiting BKCa channels with paxilline suppressed both spontaneous and LPS-stimulated uterine contractions. Furthermore, application of BKCa inhibitors (paxilline or iberiotoxin) after LPS inhibited BKCa channel activity in mMSMCs. Moreover, pretreatment with BKCa inhibitor or the TLR4 inhibitor suppressed LPS-activated BKCa currents. Our study demonstrates that BKCa channels are involved in both basal and LPS-stimulated uterine contraction in pregnant mice.

CITED2 is a conserved regulator of the uterine-placental interface.

Establishment of the hemochorial uterine-placental interface requires exodus of trophoblast cells from the placenta and their transformative actions on the uterus, which represent processes critical for a successful pregnancy, but are poorly understood. We examined the involvement of CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 2 (CITED2) in rat and human trophoblast cell development. The rat and human exhibit deep hemochorial placentation. CITED2 was distinctively expressed in the junctional zone (JZ) and invasive trophoblast cells of the rat. Homozygous Cited2 gene deletion resulted in placental and fetal growth restriction. Small Cited2 null placentas were characterized by disruptions in the JZ, delays in intrauterine trophoblast cell invasion, and compromised plasticity. In the human placentation site, CITED2 was uniquely expressed in the extravillous trophoblast (EVT) cell column and importantly contributed to the development of the EVT cell lineage. We conclude that CITED2 is a conserved regulator of deep hemochorial placentation.

Imaging Mass Spectrometry Reveals Alterations in N-Linked Glycosylation That Are Associated With Histopathological Changes in Nonalcoholic Steatohepatitis in Mouse and Human.

Nonalcoholic steatohepatitis (NASH) is the progressive form of nonalcoholic fatty liver disease (NAFLD) and is characterized by inflammation, hepatocyte injury, and fibrosis. Further, NASH is a risk factor for cirrhosis and hepatocellular carcinoma. Previous research demonstrated that serum N-glycan profiles can be altered in NASH patients. Here, we hypothesized that these N-glycan modifications may be associated with specific liver damage in NAFLD and NASH. To investigate the N-glycome profile in tissue, imaging mass spectrometry was used for a qualitative and quantitative in situ N-linked glycan analysis of mouse and human NAFLD/NASH tissue. A murine model was used to induce NAFLD and NASH through ad libitum feeding with either a high-fat diet or a Western diet, respectively. Mice fed a high-fat diet or Western diet developed inflammation, steatosis, and fibrosis, consistent with NAFLD/NASH phenotypes. Induction of NAFLD/NASH for 18 months using high caloric diets resulted in increased expression of mannose, complex/fucosylated, and hybrid N-glycan structures compared to control mouse livers. To validate the animal results, liver biopsy specimens from 51 human NAFLD/NASH patients representing the full range of NASH Clinical Research Network fibrosis stages were analyzed. Importantly, the same glycan alterations observed in mouse models were observed in human NASH biopsies and correlated with the degree of fibrosis. In addition, spatial glycan alterations were localized specifically to histopathological changes in tissue like fibrotic and fatty areas. We demonstrate that the use of standard staining's combined with imaging mass spectrometry provide a full profile of the origin of N-glycan modifications within the tissue. These results indicate that the spatial distribution of abundances of released N-glycans correlate with regions of tissue steatosis associated with NAFLD/NASH.

Blocking the BKCa channel induces NF-κB nuclear translocation by increasing nuclear calcium concentration†.

Nuclear factor kappa B (NF-κB) transcriptionally regulates several genes involved in initiating uterine contractions. A key factor controlling NF-κB activity is its translocation to the nucleus. In myometrial smooth muscle cells (MSMCs), this translocation can be stimulated by the inflammatory molecule lipopolysaccharide (LPS) or by blocking the potassium calcium-activated channel subfamily M alpha 1 (KCNMA1 or BKCa) with paxilline (PAX). Here, we sought to determine the mechanism by which blocking BKCa causes NF-κB-p65 translocation to the nucleus in MSMCs. We show that LPS- and PAX-induced NF-κB-p65 translocation are similar in that neither depends on several mitogen-activated protein kinase pathways, but both require increased intracellular calcium (Ca2+). However, the nuclear transport inhibitor wheat germ agglutinin prevented NF-κB-p65 nuclear translocation in response to LPS but not in response to PAX. Blocking BKCa located on the plasma membrane resulted in a transient NF-κB-p65 nuclear translocation that was not sufficient to induce expression of its transcriptional target, suggesting a role for intracellular BKCa. We report that BKCa also localizes to the nucleus and that blocking nuclear BKCa results in an increase in nuclear Ca2+ in MSMCs. Together, these data suggest that BKCa localized on the nuclear membrane plays a key role in regulating nuclear Ca2+ and NF-κB-p65 nuclear translocation in MSMCs.

A rare missense variant in the ATP2C2 gene is associated with language impairment and related measures.

At least 5% of children present unexpected difficulties in expressing and understanding spoken language. This condition is highly heritable and often co-occurs with other neurodevelopmental disorders such as dyslexia and ADHD. Through an exome sequencing analysis, we identified a rare missense variant (chr16:84405221, GRCh38.p12) in the ATP2C2 gene. ATP2C2 was implicated in language disorders by linkage and association studies, and exactly the same variant was reported previously in a different exome sequencing study for language impairment (LI). We followed up this finding by genotyping the mutation in cohorts selected for LI and comorbid disorders. We found that the variant had a higher frequency in LI cases (1.8%, N = 360) compared with cohorts selected for dyslexia (0.8%, N = 520) and ADHD (0.7%, N = 150), which presented frequencies comparable to reference databases (0.9%, N = 24 046 gnomAD controls). Additionally, we observed that carriers of the rare variant identified from a general population cohort (N = 42, ALSPAC cohort) presented, as a group, lower scores on a range of reading and language-related measures compared to controls (N = 1825; minimum P = 0.002 for non-word reading). ATP2C2 encodes for an ATPase (SPCA2) that transports calcium and manganese ions into the Golgi lumen. Our functional characterization suggested that the rare variant influences the ATPase activity of SPCA2. Thus, our results further support the role of ATP2C2 locus in language-related phenotypes and pinpoint the possible effects of a specific rare variant at molecular level.

Investigating the Presence of the History of Medicine in North American Medical Education: Can One of the Medical Humanities Concisely Integrate with Biomedical and Clinical Content with Reference to Clinical Competencies?

INTRODUCTION: As a curricular topic, the history of medicine can reinforce medical knowledge while integrating the benefits associated with humanities education. This study examined the degree to which medical history was present in the curricula of North American Medical Schools, how those sessions (if any) were delivered, and who delivered them. We hypothesized that medical history would be present but that very few American Medical schools would have mandatory sessions, that the presentation of this content would be very diverse, and that sessions would be presented by a variety of professionals. METHODS: We compiled a contact list of deans and curricular administrators at each institution and recruited respondents from an online discussion board. Respondents completed a short survey regarding the curriculum at their home institution. RESULTS: We received responses from 96 of 209 institutions (46.0%). Of these, 29.2% of respondents indicated that their institution had mandatory sessions on medical history, 42.7% had non-mandatory curricular sessions, 49.0% had extracurricular sessions, and 21.4% had electives on the history of medicine. Lecture was the primary means of delivery in all modalities except for the electives, which primarily used small group discussion. Clinical faculty were the most common people overseeing these sessions, but a wide variety of others also contributed. DISCUSSION: While few North American medical schools had mandatory sessions on the history of medicine, the topic was present in very diverse ways. The lack of standardization in teaching the history of medicine may provide fertile ground for subsequent research in medical education.

The broken cycle: E2F dysfunction in cancer.

The cyclin-dependent kinase (CDK)-RB-E2F axis forms the core transcriptional machinery driving cell cycle progression, dictating the timing and fidelity of genome replication and ensuring genetic material is accurately passed through each cell division cycle. The ultimate effectors of this axis are members of a family of eight distinct E2F genes encoding transcriptional activators and repressors. E2F transcriptional activity is tightly regulated throughout the cell cycle via transcriptional and translational regulation, post-translational modifications, protein degradation, binding to cofactors and subcellular localization. Alterations in one or more key components of this axis (CDKs, cyclins, CDK inhibitors and the RB family of proteins) occur in virtually all cancers and result in heightened oncogenic E2F activity, leading to uncontrolled proliferation. In this Review, we discuss the activities of E2F proteins with an emphasis on the newest atypical E2F family members, the specific and redundant functions of E2F proteins, how misexpression of E2F transcriptional targets promotes cancer and both current and developing therapeutic strategies being used to target this oncogenic pathway.

A case-control genome-wide association study of ADHD discovers a novel association with the tenascin R (TNR) gene.

It is well-established that there is a strong genetic contribution to the aetiology of attention deficit hyperactivity disorder (ADHD). Here, we employed a hypothesis-free genome-wide association study (GWAS) design in a sample of 480 clinical childhood ADHD cases and 1208 controls to search for novel genetic risk loci for ADHD. DNA was genotyped using Illumina's Human Infinium PsychArray-24v1.2., and the data were subsequently imputed to the 1000 Genomes reference panel. Rigorous quality control and pruning of genotypes at both individual subject and single nucleotide polymorphism (SNP) levels was performed. Polygenic risk score (PGRS) analysis revealed that ADHD case-control status was explained by genetic risk for ADHD, but no other major psychiatric disorders. Logistic regression analysis was performed genome-wide to test the association between SNPs and ADHD case-control status. We observed a genome-wide significant association (p = 3.15E-08) between ADHD and rs6686722, mapped to the Tenascin R (TNR) gene. Members of this gene family are extracellular matrix glycoproteins that play a role in neural cell adhesion and neurite outgrowth. Suggestive evidence of associations with ADHD was observed for an additional 111 SNPs (⩽9.91E-05). Although intriguing, the association between DNA variation in the TNR gene and ADHD should be viewed as preliminary given the small sample size of this discovery dataset.

Endoreduplication of the mouse genome in the absence of ORC1.

The largest subunit of the origin recognition complex (ORC1) is essential for assembly of the prereplicative complex, firing of DNA replication origins, and faithful duplication of the genome. Here, we generated knock-in mice with LoxP sites flanking exons encoding the critical ATPase domain of ORC1. Global or tissue-specific ablation of ORC1 function in mouse embryo fibroblasts and fetal and adult diploid tissues blocked DNA replication, cell lineage expansion, and organ development. Remarkably, ORC1 ablation in extraembryonic trophoblasts and hepatocytes, two polyploid cell types in mice, failed to impede genome endoreduplication and organ development and function. Thus, ORC1 in mice is essential for mitotic cell divisions but dispensable for endoreduplication. We propose that DNA replication of mammalian polyploid genomes uses a distinct ORC1-independent mechanism.

Dietary fat/cholesterol-sensitive PKCß-RB signaling: Potential role in NASH/HCC axis.

Hepatocellular carcinoma (HCC) is a frequent form of cancer with a poor prognosis, and environmental factors significantly contribute to the risk. Despite knowledge that a Western-style diet is a risk factor in the development of nonalcoholic steatohepatitis (NASH) and subsequent progression to HCC, diet-induced signaling changes are not well understood. Understanding molecular mechanisms altered by diet is crucial for developing preventive and therapeutic strategies. We have previously shown that diets enriched with high-fat and high-cholesterol, shown to produce NASH and HCC, induce hepatic protein kinase C beta (PKCß) expression in mice, and a systemic loss of PKCß promotes hepatic cholesterol accumulation in response to this diet. Here, we sought to determine how PKCß and diet functionally interact during the pathogenesis of NASH and how it may promote hepatic carcinogenesis. We found that diet-induced hepatic PKCß expression is accompanied by an increase in phosphorylation of Ser780 of retinoblastoma (RB) protein. Intriguingly, PKCß-/- livers exhibited reduced RB protein levels despite increased transcription of the RB gene. It is also accompanied by reduced RBL-1 with no significant effect on RBL-2 protein levels. We also found reduced expression of the PKCß in HCC compared to non-tumorous liver in human patients. These results raise an interesting possibility that diet-induced PKCß activation represents an important mediator in the functional wiring of cholesterol metabolism and tumorigenesis through modulating stability of cell cycle-associated proteins. The potential role of PKCß in the suppression of tumorigenesis is discussed.

Dosage-dependent copy number gains in E2f1 and E2f3 drive hepatocellular carcinoma.

Disruption of the retinoblastoma (RB) tumor suppressor pathway, either through genetic mutation of upstream regulatory components or mutation of RB1 itself, is believed to be a required event in cancer. However, genetic alterations in the RB-regulated E2F family of transcription factors are infrequent, casting doubt on a direct role for E2Fs in driving cancer. In this work, a mutation analysis of human cancer revealed subtle but impactful copy number gains in E2F1 and E2F3 in hepatocellular carcinoma (HCC). Using a series of loss- and gain-of-function alleles to dial E2F transcriptional output, we have shown that copy number gains in E2f1 or E2f3b resulted in dosage-dependent spontaneous HCC in mice without the involvement of additional organs. Conversely, germ-line loss of E2f1 or E2f3b, but not E2f3a, protected mice against HCC. Combinatorial mapping of chromatin occupancy and transcriptome profiling identified an E2F1- and E2F3B-driven transcriptional program that was associated with development and progression of HCC. These findings demonstrate a direct and cell-autonomous role for E2F activators in human cancer.

E2f8 mediates tumor suppression in postnatal liver development.

E2F-mediated transcriptional repression of cell cycle-dependent gene expression is critical for the control of cellular proliferation, survival, and development. E2F signaling also interacts with transcriptional programs that are downstream of genetic predictors for cancer development, including hepatocellular carcinoma (HCC). Here, we evaluated the function of the atypical repressor genes E2f7 and E2f8 in adult liver physiology. Using several loss-of-function alleles in mice, we determined that combined deletion of E2f7 and E2f8 in hepatocytes leads to HCC. Temporal-specific ablation strategies revealed that E2f8's tumor suppressor role is critical during the first 2 weeks of life, which correspond to a highly proliferative stage of postnatal liver development. Disruption of E2F8's DNA binding activity phenocopied the effects of an E2f8 null allele and led to HCC. Finally, a profile of chromatin occupancy and gene expression in young and tumor-bearing mice identified a set of shared targets for E2F7 and E2F8 whose increased expression during early postnatal liver development is associated with HCC progression in mice. Increased expression of E2F8-specific target genes was also observed in human liver biopsies from HCC patients compared to healthy patients. In summary, these studies suggest that E2F8-mediated transcriptional repression is a critical tumor suppressor mechanism during postnatal liver development.

The Spectrum of E2F in Liver Disease--Mediated Regulation in Biology and Cancer.

Uncoordinated cell growth is one of the fundamental concepts in carcinogenesis and occurs secondary to dysregulation of the cell cycle. The E2Fs are a large family of transcription factors and are key regulators of the cell cycle. The activation of E2Fs is intimately regulated by retinoblastoma 1 (RB1). The RB pathway has been implicated in almost every human malignancy. Recently there have been exciting developments in the E2F field using animal models to better understand the role of E2Fs in vivo. Genetic mouse models have proven essential in implicating E2Fs in hepatocellular carcinoma (HCC) and liver disease. In this review, the general structure and function of E2Fs as well as the role for E2Fs in the development of HCC and liver disease is evaluated. Specifically, what is known about E2Fs in human disease is explored in depth, and future directions are discussed.

Dynamic Regulation of AP-1 Transcriptional Complexes Directs Trophoblast Differentiation.

Placentation is a process that establishes the maternal-fetal interface and is required for successful pregnancy. The epithelial component of the placenta consists of trophoblast cells, which possess the capacity for multilineage differentiation and are responsible for placenta-specific functions. FOS-like antigen 1 (FOSL1), a component of AP-1 transcription factor complexes, contributes to the regulation of placental development. FOSL1 expression is restricted to trophoblast giant cells and invasive trophoblast cells. In the present study, we characterized the FOSL1 regulatory pathway in rat trophoblast cells. Transcriptome profiling in control and FOSL1 knockdown cells identified FOSL1-dependent gene sets linked to endocrine and invasive functions. FOSL1 was shown to occupy AP-1 binding sites within these gene loci, as determined by chromatin immunoprecipitation (ChIP). Complementary in vivo experiments using trophoblast-specific lentiviral delivery of FOSL1 short hairpin RNAs (shRNAs) provided in vivo validation of FOSL1 targets. FOSL1 actions require a dimerization partner. Coimmunoprecipitation, coimmunolocalization, and ChIP analyses showed that FOSL1 interacts with JUNB and, to a lesser extent, JUN in differentiating trophoblast cells. Knockdown of FOSL1 and JUNB expression inhibited both endocrine and invasive properties of trophoblast cells. In summary, FOSL1 recruits JUNB to form AP-1 transcriptional complexes that specifically regulate the endocrine and invasive trophoblast phenotypes.

On the role of NOS1 ex1f-VNTR in ADHD-allelic, subgroup, and meta-analysis.

Attention deficit/ hyperactivity disorder (ADHD) is a heritable neurodevelopmental disorder featuring complex genetics with common and rare variants contributing to disease risk. In a high proportion of cases, ADHD does not remit during adolescence but persists into adulthood. Several studies suggest that NOS1, encoding nitric oxide synthase I, producing the gaseous neurotransmitter NO, is a candidate gene for (adult) ADHD. We here extended our analysis by increasing the original sample, adding two further samples from Norway and Spain, and conducted subgroup and co-morbidity analysis. Our previous finding held true in the extended sample, and also meta-analysis demonstrated an association of NOS1 ex1f-VNTR short alleles with adult ADHD (aADHD). Association was restricted to females, as was the case in the discovery sample. Subgroup analysis on the single allele level suggested that the 21-repeat allele caused the association. Regarding subgroups, we found that NOS1 was associated with the hyperactive/impulsive ADHD subtype, but not to pure inattention. In terms of comorbidity, major depression, anxiety disorders, cluster C personality disorders and migraine were associated with short repeats, in particular the 21-repeat allele. Also, short allele carriers had significantly lower IQ. Finally, we again demonstrated an influence of the repeat on gene expression in human post-mortem brain samples. These data validate the role of NOS-I in hyperactive/impulsive phenotypes and call for further studies into the neurobiological underpinnings of this association. © 2015 Wiley Periodicals, Inc.

Identification and functional characterisation of a novel dopamine beta hydroxylase gene variant associated with attention deficit hyperactivity disorder.

OBJECTIVES: Dysregulation in neurotransmitter signalling has been implicated in the aetiology of attention deficit hyperactivity disorder (ADHD). Polymorphisms of the gene encoding dopamine beta hydroxylase (DBH) have been reported to be associated with ADHD; however, small sample sizes have led to inconsistency. METHODS: We conducted transmission disequilibrium test analysis in 794 nuclear families to examine the relationship between DBH and ADHD. The effects of the ADHD-associated polymorphisms on gene expression were assessed by luciferase reporter assays in a human neuroblastoma cell line, SH-SY5Y. RESULTS: A SNP within the 3' untranslated region of DBH rs129882 showed a significant association with ADHD (χ(2) = 9.71, p = 0.0018, OR = 1.37). This association remained significant after Bonferroni correction for multiple testing (p = 0.02). Further, allelic variation in rs129882 significantly impacted luciferase expression. Specifically, the C allele of the ADHD-associated rs129882 SNP produced a 2-fold decrease (p < 0.001) in luciferase activity. CONCLUSIONS: These data demonstrate for the first time that a DBH gene variant, rs129882, which confers risk to ADHD is also associated with reduced in vitro gene expression. Reduced DBH expression would be consistent with decreased conversion of dopamine to noradrenaline and thus with a relative hypo-noradrenergic state in ADHD.