Fibulin-4 exerts a dual role in LTBP-4L-mediated matrix assembly and function.

Elastogenesis is a hierarchical process by which cells form functional elastic fibers, providing elasticity and the ability to regulate growth factor bioavailability in tissues, including blood vessels, lung, and skin. This process requires accessory proteins, including fibulin-4 and -5, and latent TGF binding protein (LTBP)-4. Our data demonstrate mechanisms in elastogenesis, focusing on the interaction and functional interdependence between fibulin-4 and LTBP-4L and its impact on matrix deposition and function. We show that LTBP-4L is not secreted in the expected extended structure based on its domain composition, but instead adopts a compact conformation. Interaction with fibulin-4 surprisingly induced a conformational switch from the compact to an elongated LTBP-4L structure. This conversion was only induced by fibulin-4 multimers associated with increased avidity for LTBP-4L; fibulin-4 monomers were inactive. The fibulin-4-induced conformational change caused functional consequences in LTBP-4L in terms of binding to other elastogenic proteins, including fibronectin and fibrillin-1, and of LTBP-4L assembly. A transient exposure of LTBP-4L with fibulin-4 was sufficient to stably induce conformational and functional changes; a stable complex was not required. These data define fibulin-4 as a molecular extracellular chaperone for LTBP-4L. The altered LTBP-4L conformation also promoted elastogenesis, but only in the presence of fibulin-4, which is required to escort tropoelastin onto the extended LTBP-4L molecule. Altogether, this study provides a dual mechanism for fibulin-4 in 1) inducing a stable conformational and functional change in LTBP-4L, and 2) promoting deposition of tropoelastin onto the elongated LTBP-4L.

Mutations in NALCN cause an autosomal-recessive syndrome with severe hypotonia, speech impairment, and cognitive delay.

Sodium leak channel, nonselective (NALCN) is a voltage-independent and cation-nonselective channel that is mainly responsible for the leaky sodium transport across neuronal membranes and controls neuronal excitability. Although NALCN variants have been conflictingly reported to be in linkage disequilibrium with schizophrenia and bipolar disorder, to our knowledge, no mutations have been reported to date for any inherited disorders. Using linkage, SNP-based homozygosity mapping, targeted sequencing, and confirmatory exome sequencing, we identified two mutations, one missense and one nonsense, in NALCN in two unrelated families. The mutations cause an autosomal-recessive syndrome characterized by subtle facial dysmorphism, variable degrees of hypotonia, speech impairment, chronic constipation, and intellectual disability. Furthermore, one of the families pursued preimplantation genetic diagnosis on the basis of the results from this study, and the mother recently delivered healthy twins, a boy and a girl, with no symptoms of hypotonia, which was present in all the affected children at birth. Hence, the two families we describe here represent instances of loss of function in human NALCN.

PAC down-regulates estrogen receptor alpha and suppresses epithelial-to-mesenchymal transition in breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive histological subtype with limited treatment options and very poor prognosis following progression after standard chemotherapeutic regimens. Therefore, novel molecules and therapeutic options are urgently needed for this category of patients. Recently, we have identified PAC as a curcumin analogue with potent anti-cancer features. METHODS: HPLC was used to evaluate the stability of PAC and curcumin in PBS and also in circulating blood. Cytotoxicity/apoptosis was assessed in different breast cancer cell lines using propidium iodide/annexinV associated with flow cytometry. Furthermore, immunoblotting analysis determined the effects of PAC on different oncogenic proteins and pathways. Additionally, the real time xCELLigence RTCA technology was applied to investigate the effect of PAC on the cellular proliferation, migration and invasion capacities. RESULTS: PAC is more stable than curcumin in PBS and in circulating blood. Furthermore, we have shown differential sensitivity of estrogen receptor-alfa positive (ERα(+)) and estrogen receptor alfa negative (ERα(-)) breast cancer cells to PAC, which down-regulated ERα in both cell types. This led to complete disappearance of ERα in ERα(-) cells, which express very low level of this receptor. Interestingly, specific down-regulation of ERα in receptor positive cells increased the apoptotic response of these cells to PAC, confirming that ERα inhibits PAC-dependent induction of apoptosis, which could be mediated through ERα down-regulation. Additionally, PAC inhibited the proliferation and suppressed the epithelial-to-mesenchymal transition process in breast cancer cells, with higher efficiency on the TNBC subtype. This effect was also observed in vivo on tumor xenografts. Additionally, PAC suppressed the expression/secretion of 2 important cytokines IL-6 and MCP-1, and consequently inhibited the paracrine procarcinogenic effects of breast cancer cells on breast stromal fibroblasts. CONCLUSION: These results indicate that PAC could be considered as important candidate for future therapeutic options against the devastating TNBC subtype.

PAD2-mediated citrullination of Fibulin-5 promotes elastogenesis.

The formation of elastic fibers is active only in the perinatal period. How elastogenesis is developmentally regulated is not fully understood. Citrullination is a unique form of post-translational modification catalyzed by peptidylarginine deiminases (PADs), including PAD1-4. Its physiological role is largely unknown. By using an unbiased proteomic approach of lung tissues, we discovered that FBLN5 and LTBP4, two key elastogenic proteins, were temporally modified in mouse and human lungs. We further demonstrated that PAD2 citrullinated FBLN5 preferentially in young lungs compared to adult lungs. Genetic ablation of PAD2 resulted in attenuated elastogenesis in vitro and age-dependent emphysema in vivo. Mechanistically, citrullination protected FBLN5 from proteolysis and subsequent inactivation of its elastogenic activity. Furthermore, citrullinated but not native FBLN5 partially rescued in vitro elastogenesis in the absence of PAD activity. Our data uncover a novel function of citrullination, namely promoting elastogenesis, and provide additional insights to how elastogenesis is regulated.

Venom of the desert black snake Walterinnesia aegyptia enhances anti-tumor immunity via its beneficial modulatory effects on pro- and anti-tumorigenic inflammatory mediators in cultured colon cancer cells.

The role of inflammation in colon cancer is understood as a well-accepted factor that has the tendency to release multiple pro- and anti-tumorigenic inflammatory mediators. Inflammation-induced increased expression of anti-tumorigenic inflammatory mediators and decreased expression of pro-tumorigenic inflammatory mediators encourage beneficial inflammatory effects in terms of powerful anti-tumor immunity. The present study aims to screen the beneficial inflammatory effects of Walterinnesia aegyptia venom via determining its modulatory tendency on the expression of 40 pro- and anti-tumorigenic inflammatory mediators (cytokines/growth factors/chemokines) in LoVo human colon cancer cell line. LoVo-cells were treated with varying doses of crude venom of W. aegyptia. Cell viability was checked utilizing flow cytometry, and IC50 of venom was determined. Venom-induced inflammatory effects were evaluated on the expression of 40 different inflammatory mediators (12 anti-tumorigenic cytokines, 11 pro-tumorigenic cytokines, 7 pro-tumorigenic growth factors, 9 pro-tumorigenic chemokines and 1 anti-tumorigenic chemokine) in treated LoVo-cells [utilizing enzyme-linked immunosorbent assay (ELISA)] and compared with controls. Treatment of venom induced significant cytotoxic effects on inflamed LoVo-cells. IC50 treatment of venom caused significant modulations on the expression of 22 inflammatory mediators in treated LoVo-cells. The beneficial modulatory effects of venom were screened via its capability to significantly increase the expression of five powerful anti-tumorigenic mediators (IL-9, IL-12p40, IL-15, IL-1RA and Fractalkine) and decrease the expression of four major pro-tumorigenic mediators (IL-1ß, VEGF, MCP-1 and MCP-3). Walterinnesia aegyptia venom-induced beneficial modulations on the expression of nine crucial pro/anti-tumorigenic inflammatory mediators can be effectively used to enhance powerful anti-tumor immunity against colon cancer.

Identification of novel genomic imbalances in Saudi patients with congenital heart disease.

BACKGROUND: Quick genetic diagnosis of a patient with congenital heart disease (CHD) is quite important for proper health care and management. Copy number variations (CNV), chromosomal imbalances and rearrangements have been frequently associated with CHD. Previously, due to limitations of microscope based standard karyotyping techniques copious CNVs and submicroscopic imbalances could not be detected in numerous CHD patients. The aim of our study is to identify cytogenetic abnormalities among the selected CHD cases (n = 17) of the cohort using high density oligo arrays. RESULTS: Our screening study indicated that six patients (~35%) have various cytogenetic abnormalities. Among the patients, only patient 2 had a duplication whereas the rest carried various deletions. The patients 1, 4 and 6 have only single large deletions throughout their genome; a 3.2 Mb deletion on chromosome 7, a 3.35 Mb deletion on chromosome 3, and a 2.78 Mb a deletion on chromosome 2, respectively. Patients 3 and 5 have two deletions on different chromosomes. Patient 3 has deletions on chromosome 2 (2q24.1; 249 kb) and 16 (16q22.2; 1.8 Mb). Patient 4 has a 3.35 Mb an interstitial deletion on chromosome 3 (3q13.2q13.31).Based on our search on the latest available literature, our study is the first inclusive array CGH evaluation on Saudi cohort of CHD patients. CONCLUSIONS: This study emphasizes the importance of the arrays in genetic diagnosis of CHD. Based on our results the high resolution arrays should be utilized as first-tier diagnostic tool in clinical care as suggested before by others. Moreover, previously evaluated negative CHD cases (based on standard karyotyping methods) should be re-examined by microarray based cytogenetic methods.