Enhanced Ca(2+) signaling, mild primary aldosteronism, and hypertension in a familial hyperaldosteronism mouse model (Cacna1h(M1560V/+))

Gain-of-function mutations in the CACNA1H gene (encoding the T-type calcium channel Ca(V)3.2) cause autosomal-dominant familial hyperaldosteronism type IV (FH-IV) and early-onset hypertension in humans. We used CRISPR/Cas9 to generate Cacna1h(M1560V/+) knockin mice as a model of the most common FH-IV mutation, along with corresponding knockout mice (Cacna1h(-/-)). Adrenal morphology of both Cacna1h(M1560V/+) and Cacna1h(-/-) mice was normal. Cacna1h(M1560V/+) mice had elevated aldosterone:renin ratios (a screening parameter for primary aldosteronism). Their adrenal Cyp11b2 (aldosterone synthase) expression was increased and remained elevated on a high-salt diet (relative autonomy, characteristic of primary aldosteronism), but plasma aldosterone was only elevated in male animals. The systolic blood pressure of Cacna1h(M1560V/+) mice was 8 mmHg higher than in wild-type littermates and remained elevated on a high-salt diet. Cacna1h(-/-) mice had elevated renal Ren1 (renin-1) expression but normal adrenal Cyp11b2 levels, suggesting that in the absence of Ca(V)3.2, stimulation of the renin-angiotensin system activates alternative calcium entry pathways to maintain normal aldosterone production. On a cellular level, Cacna1h(M1560V/+) adrenal slices showed increased baseline and peak intracellular calcium concentrations in the zona glomerulosa compared to controls, but the frequency of calcium spikes did not rise. We conclude that FH-IV, on a molecular level, is caused by elevated intracellular Ca(2+) concentrations as a signal for aldosterone production in adrenal glomerulosa cells. We demonstrate that a germline Cacna1h gain-of-function mutation is sufficient to cause mild primary aldosteronism, whereas loss of Ca(V)3.2 channel function can be compensated for in a chronic setting

GAS41 interacts with transcription factor AP-2β and stimulates AP-2β-mediated transactivation

Transcription factor AP-2 regulates transcription of a number of genes involving mammalian development, differentiation and carcinogenesis. Recent studies have shown that interaction partners can modulate the transcriptional activity of AP-2 over the downstream targets. In this study, we reported the identification of GAS41 as an interaction partner of AP-2β. We documented the interaction both in vivo by co-immunoprecipitation as well as in vitro through glutathione S-transferase (GST) pull-down assays. We also showed that the two proteins are co-localized in the nuclei of mammalian cells. We further mapped the interaction domains between the two proteins to the C-termini of both AP-2β and GAS41, respectively. Furthermore, we have identified three critical residues of GAS41 that are important for the interaction between the two proteins. In addition, by transient co-expression experiments using reporter containing three AP-2 consensus binding sites in the promoter region, we found that GAS41 stimulates the transcriptional activity of AP-2β over the reporter. Finally, electrophoretic mobility shift assay (EMSA) suggested that GAS41 enhances the DNA-binding activity of AP-2β. Our data provide evidence for a novel cellular function of GAS41 as a transcriptional co-activator for AP-2β

Genes encoding critical transcriptional activators for murine neural tube development and human spina bifida: a case-control study

<p>Abstract</p> <p>Background</p> <p>Spina bifida is a malformation of the neural tube and is the most common of neural tube defects (NTDs). The etiology of spina bifida is largely unknown, although it is thought to be multi-factorial, involving multiple interacting genes and environmental factors. Mutations in transcriptional co-activator genes-<it>Cited2</it>, <it>p300</it>, <it>Cbp</it>, <it>Tfap2α</it>, <it>Carm1 </it>and <it>Cart1 </it>result in NTDs in murine models, thus prompt us to investigate whether homologues of these genes are associated with NTDs in humans.</p> <p>Methods</p> <p>Data and biological samples from 297 spina bifida cases and 300 controls were derived from a population-based case-control study conducted in California. 37 SNPs within <it>CITED2</it>, <it>EP300</it>, <it>CREBBP</it>, <it>TFAP2A</it>, <it>CARM1 </it>and <it>ALX1 </it>were genotyped using an ABI SNPlex assay. Odds ratios and 95% confidence intervals were calculated for alleles, genotypes and haplotypes to evaluate the risk for spina bifida.</p> <p>Results</p> <p>Several SNPs showed increased or decreased risk, including <it>CITED2 </it>rs1131431 (OR = 5.32, 1.04~27.30), <it>EP300 </it>rs4820428 (OR = 1.30, 1.01~1.67), <it>EP300 </it>rs4820429 (OR = 0.50, 0.26~0.50, in whites, OR = 0.7, 0.49~0.99 in all subjects), <it>EP300 </it>rs17002284 (OR = 0.43, 0.22~0.84), <it>TFAP2A </it>rs3798691 (OR = 1.78, 1.13~2.87 in Hispanics), <it>CREBBP </it>rs129986 (OR = 0.27, 0.11~0.69), <it>CARM1 </it>rs17616105 (OR = 0.41, 0.22~0.72 in whites). In addition, one haplotype block in <it>EP300 </it>and one in <it>TFAP2A </it>appeared to be associated with increased risk.</p> <p>Conclusions</p> <p>Modest associations were observed in <it>CITED2</it>, <it>EP300</it>, <it>CREBBP</it>, <it>TFAP2A </it>and <it>CARM1 </it>but not <it>ALX1</it>. However, these modest associations were not statistically significant after correction for multiple comparisons. Searching for potential functional variants and rare causal mutations is warranted in these genes.</p

A Heart-Hand Syndrome Gene: Tfap2b Plays a Critical Role in the Development and Remodeling of Mouse Ductus Arteriosus and Limb Patterning

BACKGROUND: Patent ductus arteriosus (PDA) is one of the most common forms of congenital heart disease. Mutations in transcription factor TFAP2B cause Char syndrome, a human disorder characterized by PDA, facial dysmorphysm and hand anomalies. Animal research data are needed to understand the mechanisms. The aim of our study was to elucidate the pathogenesis of Char syndrome at the molecular level. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression of Tfap2b during mouse development was studied, and newborns of Tfap2b-deficient mice were examined to identify phenotypes. Gel shift assays had been carried out to search for Tfap2 downstream genes. Promoters of candidate genes were cloned into a reporter construct and used to demonstrate their regulation by Tfap2b in cell transfection. In situ hybridizations showed that the murine transcription factor Tfap2b was expressed during the entire development of mouse ductus arteriosus. Histological examination of ductus arteriosus from Tfap2b knockout mice 6 hours after birth revealed that they were not closed. Consequently, the lungs of Tfap2b(-/-) mice demonstrated progressive congestion of the pulmonary capillaries, which was postulated to result secondarily from PDA. In addition, Tfap2b was expressed in the limb buds, particularly in the posterior limb field during development. Lack of Tfap2b resulted in bilateral postaxial accessory digits. Further study indicated that expressions of bone morphogenetic protein (Bmp) genes, which are reported to be involved in the limb patterning and ductal development, were altered in limb buds of Tfap2b-deficient embryos, due to direct control of Bmp2 and Bmp4 promoter activity by Tfap2b. CONCLUSIONS/SIGNIFICANCE: Tfap2b plays important roles in the development of mouse ductus arteriosus and limb patterning. Loss of Tfap2b results in altered Bmp expression that may cause the heart-limb defects observed in Tfap2b mouse mutants and Char syndrome patients. The Tfap2b knockout mouse may add to the very limited available animal models of PDA

Identification of target genes of transcription factor activator protein 2 gamma in breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Activator protein 2 gamma (AP-2γ) is a member of the transcription factor activator protein-2 (AP-2) family, which is developmentally regulated and plays a role in human neoplasia. AP-2γ has been found to be overexpressed in most breast cancers, and have a dual role to inhibit tumor initiation and promote tumor progression afterwards during mammary tumorigensis.</p> <p>Methods</p> <p>To identify the gene targets that mediate its effects, we performed chromatin immunoprecipitation (ChIP) to isolate AP-2γ binding sites on genomic DNA from human breast cancer cell line MDA-MB-453.</p> <p>Results</p> <p>20 novel DNA fragments proximal to potential AP-2γ targets were obtained. They are categorized into functional groups of carcinogenesis, metabolism and others. A combination of sequence analysis, reporter gene assays, quantitative real-time PCR, electrophoretic gel mobility shift assays and immunoblot analysis further confirmed the four AP-2γ target genes in carcinogenesis group: ErbB2, CDH2, HPSE and IGSF11. Our results were consistent with the previous reports that ErbB2 was the target gene of AP-2γ. Decreased expression and overexpression of AP-2γ in human breast cancer cells significantly altered the expression of these four genes, indicating that AP-2γ directly regulates them.</p> <p>Conclusion</p> <p>This suggested that AP-2γ can coordinate the expression of a network of genes, involving in carcinogenesis, especially in breast cancer. They could serve as therapeutic targets against breast cancers in the future.</p

Ablation of Mrds1/Ofcc1 Induces Hyper-γ-Glutamyl Transpeptidasemia without Abnormal Head Development and Schizophrenia-Relevant Behaviors in Mice

Mutations in the Opo gene result in eye malformation in medaka fish. The human ortholog of this gene, MRDS1/OFCC1, is a potentially causal gene for orofacial cleft, as well as a susceptibility gene for schizophrenia, a devastating mental illness. Based on this evidence, we hypothesized that this gene could perform crucial functions in the development of head and brain structures in vertebrates. To test this hypothesis, we created Mrds1/Ofcc1-null mice. Mice were examined thoroughly using an abnormality screening system referred to as “the Japan Mouse Clinic”. No malformations of the head structure, eye or other parts of the body were apparent in these knockout mice. However, the mutant mice showed a marked increase in serum γ-glutamyl transpeptidase (GGT), a marker for liver damage, but no abnormalities in other liver-related measurements. We also performed a family-based association study on the gene in schizophrenia samples of Japanese origin. We found five single nucleotide polymorphisms (SNPs) located across the gene that showed significant transmission distortion, supporting a prior report of association in a Caucasian cohort. However, the knockout mice showed no behavioral phenotypes relevant to schizophrenia. In conclusion, disruption of the Mrds1/Ofcc1 gene elicits asymptomatic hyper-γ-glutamyl-transpeptidasemia in mice. However, there were no phenotypes to support a role for the gene in the development of eye and craniofacial structures in vertebrates. These results prompt further examination of the gene, including its putative contribution to hyper-γ-glutamyl transpeptidasemia and schizophrenia

Endovascular Abdominal Aortic Aneurysm Repair With Ovation Alto Stent Graft: Protocol for the ALTAIR (ALTo endogrAft Italian Registry) Study

Background: Since 2010, the Ovation Abdominal Stent Graft System has offered an innovative sealing option for abdominal aortic aneurysm (AAA) by including a sealing ring filled with polymer 13 mm from the renal arteries. In August 2020, the redesigned Ovation Alto, with a sealing ring 6 mm closer to the top of the fabric, received CE Mark approval. Objective: This registry study aims to evaluate intraoperative, perioperative, and postoperative results in patients treated by the Alto stent graft (Endologix Inc.) for elective AAA repair in a multicentric consecutive experience. Methods: All consecutive eligible patients submitted to endovascular aneurysm repair (EVAR) by Alto Endovascular AAA implantation will be included in this analysis. Patients will be submitted to EVAR procedures based on their own preferences, anatomical features, and operators experience. An estimated number of 300 patients submitted to EVAR with Alto stent graft should be enrolled. It is estimated that the inclusion period will be 24 months. The follow-up period is set to be 5 years. Full data sets and cross-sectional images of contrast-enhanced computed tomography scan performed before EVAR, at the first postoperative month, at 24 or 36 months, and at 5-year follow-up interval will be reported in the central database for a centralized core laboratory review of morphological changes. The primary endpoint of the study is to evaluate the technical and clinical success of EVAR with the Alto stent graft in short- (90-day), mid- (1-year), and long-term (5-year) follow-up periods. The following secondary endpoints will be also addressed: operative time; intraoperative radiation exposure; contrast medium usage; AAA sac shrinkage at 12-month and 5-year follow-up; any potential role of patients' baseline characteristics, valuated on preoperative computed tomography angiographic study, and of device configuration (number of component) in the primary endpoint. Results: The study is currently in the recruitment phase and the final patient is expected to be treated by the end of 2023 and then followed up for 5 years. A total of 300 patients will be recruited. Analyses will focus on primary and secondary endpoints. Updated results will be shared at 1- and 3-5-year follow-ups. Conclusions: The results from this registry study could validate the safety and effectiveness of the new design of the Ovation Alto Stent Graft. The technical modifications to the endograft could allow for accommodation of a more comprehensive range of anatomies on-label

A black-box, general purpose quadratic self-consistent field code with and without Cholesky decomposition of the two-electron integrals

We present the implementation of a quadratically convergent self-consistent field (QCSCF) algorithm based on an adaptive trust-radius optimisation scheme for restricted open-shell Hartree–Fock (ROHF), restricted Hartree–Fock (RHF), and unrestricted Hartree–Fock (UHF) references. The algorithm can exploit Cholesky decomposition (CD) of the two-electron integrals to allow calculations on larger systems. The most important feature of the QCSCF code lies in its black-box nature–probably the most important quality desired by a generic user. As shown for pilot applications, it does not require one to tune the self-consistent field (SCF) parameters (damping, Pulay's DIIS, and other similar techniques) in difficult-to-converge molecules. Also, it can be used to obtain a very tight convergence with extended basis sets–a situation often needed when computing high-order molecular properties–where the standard SCF algorithm starts to oscillate. Nevertheless, trouble may appear even with a QCSCF solver. In this respect, we discuss what can go wrong, focusing on the multiple UHF solutions of ortho-benzyne

Serum and Glucocorticoid-Inducible Kinase 1 (SGK1): An Important Contributor to Diarrhea and Malabsorption in Microvillus Inclusion Disease (MVID)

Microvillus Inclusion Disease (MVID) is a lethal congenital diarrheal disease resulting from loss of function mutations in the actin motor myosin VB (MYO5B) that regulates apical traffic. MVID remains without treatment to reverse the severe diarrhea that leads to death. MVID diarrhea results from both increased fluid secretion and malabsorption of ions and carbohydrates in the small intestine. Serum and Glucocorticoid-inducible kinase 1 (SGK1), is a potent regulator of ion transporters including cystic fibrosis transmembrane conductance regulator (CFTR) in the intestine. Since SGK1 upregulates CFTR function in the intestine, we hypothesized that loss of SGK1 could potentially reduce MVID diarrhea by decreasing CFTR fluid secretion. Using CRISPR-Cas 9 approaches, we first generated MYO5B-floxed mice and to generate conditional MYO5B-KO (R26 ER;MYO5B ) mice, we cross bred with R26 ER mice. Tamoxifen-treated R26 ER;MYO5B mice resulted in characteristic features of human MVID including severe diarrhea, Microvillus Inclusions (MIs), defective apical traffic, depolarization of proteins, and ion transporters including CFTR, NHE3 and DRA. MYO5B-KO mice also showed increased phosphorylation of SGK1 and PDK1 in the intestine. SGK1-floxed (SGK1 )mice were crossed with R26 ER;MYO5B mice to generate conditional MYO5B/SGK1 DcKO (R26 ER;MYO5B ;SGK1 ) upon Tamoxifen induction. Surprisingly, tamoxifen treatment (3 day) of MYO5B/SGK1 DcKO mice resulted in more severe diarrhea compared to MYO5B (R26 ER;MYO5B ) mice. Immunoblots of intestinal lysates revealed decreased CFTR, increased alpha- and beta-ENaC, increased phosphorylation of PDK1, Nedd4-2 and pPKCι in MYO5B/SGK1 DcKO vs. MYO5B ScKO mice. Finally, fecal glucose loss was higher with reduced SGLT1 and GLUT2 by immunoblot in MYO5B/SGK1 DcKO vs MYO5B ScKO mice. We conclude that activation of SGK1 pathway and CFTR contribute to worsening of diarrhea and carbohydrate malabsorption in MVID

Loss of Serum Glucocorticoid-Inducible Kinase 1 SGK1 Worsens Malabsorption and Diarrhea in Microvillus Inclusion Disease (MVID)

Microvillus inclusion disease (MVID), a lethal congenital diarrheal disease, results from loss of function mutations in the apical actin motor myosin VB (MYO5B). How loss of MYO5B leads to both malabsorption and fluid secretion is not well understood. Serum glucocorticoid-inducible kinase 1 (SGK1) regulates intestinal carbohydrate and ion transporters including cystic fibrosis transmembrane conductance regulator (CFTR). We hypothesized that loss of SGK1 could reduce CFTR fluid secretion and MVID diarrhea. Using CRISPR-Cas9 approaches, we generated R26(Cre)ER;MYO5B(f/f) conditional single knockout (cMYO5BKO) and R26(Cre)ER;MYO5B(f/f);SGK1(f/f) double knockout (cSGK1/MYO5B-DKO) mice. Tamoxifen-treated cMYO5BKO mice resulted in characteristic features of human MVID including severe diarrhea, microvillus inclusions (MIs) in enterocytes, defective apical traffic, and depolarization of transporters. However, apical CFTR distribution was preserved in crypts and depolarized in villus enterocytes, and CFTR high expresser (CHE) cells were observed. cMYO5BKO mice displayed increased phosphorylation of SGK1, PDK1, and the PDK1 target PKC iota in the intestine. Surprisingly, tamoxifen-treated cSGK1/MYO5B-DKO mice displayed more severe diarrhea than cMYO5BKO, with preservation of apical CFTR and CHE cells, greater fecal glucose and reduced SGLT1 and GLUT2 in the intestine. We conclude that loss of SGK1 worsens carbohydrate malabsorption and diarrhea in MVID