Prevalence and clinical significance of co-existing mutations in MED12 and FH in uterine fibroids of Australian women.

Uterine fibroids are exceedingly common benign tumours of the female reproductive system and cause severe symptoms, including acute pain, bleeding, and infertility. Fibroids are frequently associated with genetic alterations affecting mediator complex subunit 12 (MED12), fumarate hydratase (FH), high mobility group AT-hook 2 (HMGA2) and collagen, type IV alpha 5 and alpha 6 (COL4A5-COL4A6). Recently, we reported MED12 exon 2 mutations in 39 out of 65 uterine fibroids (60%) from 14 Australian patients. The aim of this study was to evaluate the status of FH mutations in MED12 mutation-positive and mutation-negative uterine fibroids. FH mutation screening of altogether 65 uterine fibroids and corresponding adjacent normal myometrium (n = 14) was carried out by Sanger sequencing. Three out of 14 patients displayed somatic mutations in FH exon 1 in addition to harbouring MED12 mutation in uterine fibroids. This study is the first to report that the mutations in MED12 and FH co-exist in uterine fibroids of Australian women.

The effect of bulk-fill composites: Activa and Smart Dentin Replacement on cuspal deflection in endodontically treated teeth with different access cavity designs.

Aim: The aim was to evaluate the effect of bulk-fill composites - Activa and Smart Dentin Replacement (SDR) on cuspal deflection in endodontically treated teeth with different access cavity designs. Materials and Methods: Forty-two maxillary first premolar teeth were randomly divided into three experimental groups based on different access designs as Group I - conservative endodontic cavities (CECs), Group II - traditional endodontic cavities, and Group III - CECs with diverging walls and two subgroups, based on the different access cavity restorations subgroup A-Activa and subgroup B-SDR with n = 7 per group. Root canal treatment was completed under the dental operating microscope. Cuspal deflection was assessed by measuring the distance between the glass beads using an optical microscope and micrometer immediately after a Root canal treatment and final measurements after access restorations. Data were statistically analyzed and tabulated. Results: Results showed that the access cavity restored with Activa showed the highest cuspal deflection than SDR in all the groups (P < 0.05). Conservative access designs showed the least cuspal deflection among other groups. Conclusion: CEC designs showed lesser cuspal deflection than traditional endodontic cavity designs. SDR can be considered as a choice of material for access restorations.

AEBP1 down regulation induced cell death pathway depends on PTEN status of glioma cells.

Glioblastoma (GBM) is the most common aggressive form of brain cancer with overall dismal prognosis (10-12 months) despite all current multimodal treatments. Previously we identified adipocyte enhancer binding protein 1 (AEBP1) as a differentially regulated gene in GBM. On probing the role of AEBP1 over expression in glioblastoma, we found that both cellular proliferation and survival were affected upon AEBP1 silencing in glioma cells, resulting in cell death. In the present study we report that the classical caspase pathway components are not activated in cell death induced by AEBP1 down regulation in PTEN-deficient (U87MG and U138MG) cells. PARP-1 was not cleaved but over-activated under AEBP1 down regulation which leads to the synthesis of PAR in the nucleus triggering the release of AIF from the mitochondria. Subsequently, AIF translocates to the nucleus along with MIF causing chromatinolysis. AEBP1 positively regulates PI3KinaseCß by the binding to AE-1 binding element in the PI3KinaseCß promoter. Loss of PI3KinaseCß expression under AEBP1 depleted condition leads to excessive DNA damage and activation of PARP-1. Furthermore, over expression of PIK3CB (in trans) in U138MG cells prevents DNA damage in these AEBP1 depleted cells. On the contrary, AEBP1 down regulation induces caspase-dependent cell death in PTEN-proficient (LN18 and LN229) cells. Ectopic expression of wild-type PTEN in PTEN-deficient U138MG cells results in the activation of canonical caspase and Akt dependent cell death. Collectively, our findings define AEBP1 as a potential oncogenic driver in glioma, with potential implications for therapeutic intervention.

Proteomic Analysis Identifies Tenascin-C Expression Is Upregulated in Uterine Fibroids.

Uterine leiomyomas (fibroids) are the most common gynecological tumors, which are enriched in the extracellular matrix (ECM). Fibroids are leading cause of abnormal uterine bleeding and hysterectomy. One of the major questions yet to be answered is the overproduction of specific ECM components in human uterine fibroids, particularly in relation to mutations in the driver gene mediator complex subunit 12 ( MED12). Surgical specimens from 14 patients with uterine leiomyoma having fibroids and corresponding adjacent normal myometrium (ANM) were utilized to analyze genetic and proteomic expression patterns in the tissue samples. MED12 mutations in the fibroids were screened by Sanger sequencing. iTRAQ was used to label the peptides in small-, medium-, and large-sized fibroid samples of annotated MED12 mutation from the same patient. The mixtures of the peptides were fractionated by hydrophilic interaction liquid chromatography (HILIC) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the differential expression proteins. Using isobaric tagged-based quantitative mass spectrometry on 3 selected patients, ECM-related protein tenascin-C (TNC) was observed significantly upregulated (>1.5-fold) with a confidence corresponding to false discovery rate (FDR) <1% in small-, medium-, and large-sized fibroid samples regardless of MED12 mutation status. The TNC was validated on additional patient samples using Western blotting (WB) and immunohistochemistry (IHC) and confirmed significant overexpression of this protein in fibroids compared to matched ANM. Proteomic analyses have identified the increased ECM protein expression, TNC, as a hallmark of uterine fibroids regardless of MED12 mutations. Further functional studies focusing on the upregulated ECM proteins in leiomyogenesis will lead to the identification of novel ECM drug targets for fibroid treatment.

Proteomic Profiling of Human Uterine Fibroids Reveals Upregulation of the Extracellular Matrix Protein Periostin.

The central characteristic of uterine fibroids is excessive deposition of extracellular matrix (ECM), which contributes to fibroid growth and bulk-type symptoms. Despite this, very little is known about patterns of ECM protein expression in fibroids and whether these are influenced by the most common genetic anomalies, which relate to MED12. We performed extensive genetic and proteomic analyses of clinically annotated fibroids and adjacent normal myometrium to identify the composition and expression patterns of ECM proteins in MED12 mutation-positive and mutation-negative uterine fibroids. Genetic sequencing of tissue samples revealed MED12 alterations in 39 of 65 fibroids (60%) from 14 patients. Using isobaric tagged-based quantitative mass spectrometry on three selected patients (n = 9 fibroids), we observed a common set of upregulated (>1.5-fold) and downregulated (<0.66-fold) proteins in small, medium, and large fibroid samples of annotated MED12 status. These two sets of upregulated and downregulated proteins were the same in all patients, regardless of variations in fibroid size and MED12 status. We then focused on one of the significant upregulated ECM proteins and confirmed the differential expression of periostin using western blotting and immunohistochemical analysis. Our study defined the proteome of uterine fibroids and identified that increased ECM protein expression, in particular periostin, is a hallmark of uterine fibroids regardless of MED12 mutation status. This study sets the foundation for further investigations to analyze the mechanisms regulating ECM overexpression and the functional role of upregulated ECM proteins in leiomyogenesis.

Ovarian hormones through Wnt signalling regulate the growth of human and mouse ovarian cancer initiating lesions.

Ovarian cancer (OC) is the most deadly gynaecological disease largely because the majority of patients are asymptomatic and diagnosed at later stages when cancer has spread to other vital organs. Therefore, the initial stages of this disease are poorly characterised. Women with BRCA1/2 mutations have a genetic predisposition for developing OC, but not all of these women develop the disease. Epidemiological findings show that lifestyle factors such as contraceptive use and pregnancy, a progesterone dominant state, decrease the risk of getting OC. How ovarian hormones modify the risk of OC is currently unclear. Our study identifies activated Wnt signalling to be a marker for precursor lesions of OC and successfully develops a mouse model that mimics the earliest events in pathogenesis of OC by constitutively activating ßcatenin. Using this model and human OC cells, we show that oestrogen promotes and progesterone suppresses the growth of OC cells.

Age related increase in mTOR activity contributes to the pathological changes in ovarian surface epithelium.

Ovarian cancer is a disease of older women. However, the molecular mechanisms of ovarian aging and their contribution to the pathogenesis of ovarian cancer are currently unclear. mTOR signalling is a major regulator of aging as suppression of this pathway extends lifespan in model organisms. Overactive mTOR signalling is present in up to 80% of ovarian cancer samples and is associated with poor prognosis. This study examined the role of mTOR signalling in age-associated changes in ovarian surface epithelium (OSE). Histological examination of ovaries from both aged mice and women revealed OSE cell hyperplasia, papillary growth and inclusion cysts. These pathological lesions expressed bonafide markers of ovarian cancer precursor lesions, Pax8 and Stathmin 1, and were presented with elevated mTOR signalling. To understand whether overactive mTOR signalling is responsible for the development of these pathological changes, we analysed ovaries of the Pten trangenic mice and found significant reduction in OSE lesions compared to controls. Furthermore, pharmacological suppression of mTOR signalling significantly decreased OSE hyperplasia in aged mice. Treatment with mTOR inhibitors reduced human ovarian cancer cell viability, proliferation and colony forming ability. Collectively, we have established the role of mTOR signalling in age-related OSE pathologies and initiation of ovarian cancer.

Synthesis and spectroscopic characterization of 3,4-difluoroacetophenone-thiosemicarbazone and its palladium(II) complex: evaluation of antimicrobial and antitumour activity.

A new cis-palladium(II)diaqua(3,4-difluoroacetophenonethiosemicarbazone complex (Pd(II) complex) is synthesized using 3,4-difluoroacetophenonethiosemicarbazone(L). The L and its Pd(II) complex are characterized and confirmed by elemental analyses, electrochemical analyses, FT-IR, FT-Raman, UV-Vis, HRMS and LC-MS techniques. Ligand L is further characterized by (1)H, (13)C and (19)F NMR spectroscopy. The crystal structure of L is unambiguously characterized by single X-ray crystallography. The ligand (L) belongs to monoclinic system with P2(1)/C space group and the unit cell parameters are a(Å)=9.1144(7), b(Å)=13.7928(7), c(Å)=8.4174(5), α(°)=90, ß(°)=100.715, γ(°)=90 and volume V(A(3))=1039.73(11). The Raman bands observed for the L and its Pd(II) complex are in good agreement with the FT-IR spectral data. The Pd(II) complex is found to be highly efficient in inhibiting the growth of human pathogens like Salmonella typhimurium and Klebsiella pneumonia with MIC value 10.0µg/mL whose inhibition zones are almost comparable with the standard antibiotic. The synthesized compounds have shown antiproliferative activity against the human breast cancer cell lines MDA-MB231 by intermitting the regular pathway of ribonucleotidereductase.

Synthesis, spectral characterization and biological activities of Mn(II) and Co(II) complexes with benzyloxybenzaldehyde-4-phenyl-3-thiosemicarbazone.

Mn(II) and Co(II) complexes of benzyloxybenzaldehyde-4-phenyl-3-thiosemicarbazone have been synthesized and characterized by the investigations of electronic and EPR spectra and X-ray diffraction. Based on the spectral studies, an octahedral geometry is assigned for the Mn(II) and Co(II) complexes. X-ray powder diffraction studies reveal that Mn(II) and Co(II) complexes have triclinic crystal lattices. The unit cell parameters of the Mn(II) complex are a=11.0469 Å, b=6.2096 Å, c=7.4145 Å, α=90.646°, ß=95.127°, γ=104.776°, V=489.7 Å(3) and those of Co(II) complex are a=9.3236 Å, b=10.2410 Å, c=7.8326 Å, α=90.694°, ß=99.694°, γ=100.476°, V=724.2 Å(3). When the free ligand and its metal complexes are subjected to antibacterial activity, the metal complexes are proved to be more active than the ligand. However with regard to in vitro antioxidant activity, the ligand exhibits greater antioxidant activity than its metal(II) complexes.

Copper(II) and nickel(II) complexes of benzyloxybenzaldehyde-4-phenyl-3-thiosemicarbazone: Synthesis, characterization and biological activity.

Benzyloxybenzaldehyde-4-phenyl-3-thiosemicarbazone ligand (L) has been synthesized from benzyloxybenzaldehyde and 4-phenyl-3-thiosemicarbazide. Complexes of this ligand with chlorides of Cu(II) and Ni(II) have been prepared. The structure of the ligand (L) is proposed based on elemental analysis, IR and (1)H NMR spectra. Its complexes with Cu(II) and Ni(II) ions are characterized from the studies of electronic as well as EPR spectra. On the basis of electronic and EPR studies, rhombically distorted octahedral structure has been proposed for Cu(II) complex while the Ni(II) complex has been found to acquire an octahedral structure. The ligand and their metal complexes have been tested in vitro for their biological effects. Their antibacterial activities against Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae) and Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) have been investigated. The prepared metal complexes exhibit higher antibacterial activities than the parent ligand. The in vitro antioxidant activity of free ligand and its metal(II) complexes have also been investigated and the results however reveal that the ligand exhibits greater antioxidant activity than its complexes.