Gonadotropin-Dependent Neuregulin-1 Signaling Regulates Female Rat Ovarian Granulosa Cell Survival.

Mammalian ovarian follicular development and maturation of an oocyte competent to be fertilized and develop into an embryo depends on tightly regulated, spatiotemporally orchestrated crosstalk among cell death, survival, and differentiation signals through extra- and intraovarian signals, as well as on a permissive ovarian follicular microenvironment. Neuregulin-1 (NRG1) is a member of the epidermal growth factor-like factor family that mediates its effects by binding to a member of the erythroblastoma (ErbB) family. Our experimental results suggest gonadotropins promote differential expression of NRG1 and erbB receptors in granulosa cells (GCs), and NRG1 in theca cells during follicular development, and promote NRG1 secretions in the follicular fluid (FF) of rat ovaries. During the estrous cycle of rat, NRG1 and erbB receptors are differentially expressed in GCs and correlate positively with serum gonadotropins and steroid hormones. Moreover, in vitro experimental studies suggest that the protein kinase C inhibitor staurosporine (STS) causes the physical destruction of GCs by the activation of caspase-3. Exogenous NRG1 treatment of GCs delayed onset of STS-induced apoptosis and inhibited cleaved caspase-3 expressions. Moreover, exogenous NRG1 treatment of GCs alters STS-induced death by maintaining the expression of ErbB2, ErbB3, pAkt, Bcl2, and BclxL proteins. Taken together, these studies demonstrate that NRG1 is gonadotropin dependent, differentially regulated in GCs and theca cells, and secreted in ovarian FF as an intracellular survival factor that may govern follicular maturation.

Expression of mitochondrial genes MT-ND1, MT-ND6, MT-CYB, MT-COI, MT-ATP6, and 12S/MT-RNR1 in colorectal adenopolyps.

Despite improvements in treatment strategies, colorectal cancer (CRC) still has high mortality rates. Most CRCs develop from adenopolyps via the adenoma-carcinoma sequence. A mechanism for inhibition of this sequence in individuals with a high risk of developing CRC is urgently needed. Differential studies of mitochondrial (mt) gene expressions in the progressive stages of CRC with villous architecture are warranted to reveal early risk assessments and new targets for chemoprevention of the disease. In the present study, reverse transcription-quantitative PCR (RT-qPCR) was used to determine the relative amount of the transcripts of six mt genes [MT-RNR1, MT-ND1, MT-COI, MT-ATP6, MT-ND6, and MT-CYB (region 648-15887)] which are involved in the normal metabolism of mitochondria. A total of 42 pairs of tissue samples obtained from colorectal adenopolyps, adenocarcinomas, and their corresponding adjacent normal tissues were examined. Additionally, electron transport chain (ETC), complexes I (NADH: ubiquinone oxidoreductase) and III (CoQH2-cytochrome C reductase), and carbonyl protein group contents were analyzed. Results indicate that there were differential expressions of the six mt genes and elevated carbonyl protein contents among the colorectal adenopolyps compared to their paired adjacent normal tissues (p < 0.05). The levels of complexes I and III were higher in tumor tissues relative to adjacent normal tissues. Noticeably, the expression of MT-COI was overexpressed in late colorectal carcinomas among all studied transcripts. Our data suggest that increased expressions in certain mt genes and elevated levels of ROS may potentially play a critical role in the colorectal tumors evolving from adenopolyps to malignant lesions.

Exosomes derived from endometriotic stromal cells have enhanced angiogenic effects in vitro.

Our objective has been to establish a pro-angiogenic role for exosomes in endometriosis and to determine whether a differential expression profile of cellular and exosomal microRNAs (miRNAs) exists in endometriosis. We performed an in vitro study of human primary endometrial stromal cells (ESCs) and human umbilical vein endothelial cells (HUVECs). We isolated and characterized exosomes from ESCs from five endometriosis patients and five phase-matched controls. Exosomes were characterized by transmission electron microscopy and NanoSight technology. MiRNA was assessed by deep sequencing and reverse transcription with quantitative polymerase chain reaction. Exosome uptake studies were achieved by means of confocal microscopy. The pro-angiogenic experiments were executed by treating HUVECs with ESC-derived exosomes. We observed differential profiles of exosomal miRNA expression between exosomes derived from endometriosis lesion cells and diseased eutopic stromal cells compared with exosomes derived from control ESCs. We also demonstrated autocrine cellular uptake of exosomes and paracrine functional angiogenic effects of exosomes on HUVECs. The results of this study support the hypothesis that exosomes derived from ESCs play autocrine/paracrine roles in the development of endometriosis, potentially modulating angiogenesis. The broader clinical implications are that Sampson's theory of retrograde menstruation possibly encompasses the finding that exosomes work as intercellular communication modulators in endometriosis.

Reactive Oxygen Species and Serous Epithelial Ovarian Adenocarcinoma.

Serous ovarian cancer (SOC) is usually diagnosed at late stage and stage-adjusted five year survival rate is low. Mortality is relatively heavy on African-Americans/Black (AA) affected with SOC compared to their Caucasian counterparts, though the cause for the disparity remains unclear. DNA damage induced by oxidative stress has been linked to ovarian cancer, but the role of oxidative stress in distinguishing differences in aggressive SOC tumors among patients is yet to be determined. This study aims to determine the levels of reactive oxygen species (ROS), malondialdehyde (MDA), reactive carbonyl groups and antioxidants in primary SOC normal, precancerous (cystadenoma, borderline) and invasive (III/IV) tissue samples obtained from AA and Caucasian subgroups. Additionally, the study seeks to investigate significant changes in the level of ROS between AA and Caucasian SOC samples. A fluorogenic probe, dichlorodihydrofluorescein (DCFH-DiOxyQ), was used to scavenge reactive oxygen species in SOC normal, precancerous and malignant stages III/IV tissue samples. Malondialdehyde (MDA), a lipid peroxidation marker, and reactive carbonyl groups were measured as indicators of oxidative injury. Moreover, antioxidant status was assessed by estimating glutathione peroxidase 3 (GPX3) enzyme levels. Results indicate ROS concentration was approximately 96% higher in the malignant tissues in comparative to the normal non-diseased controls. In addition, ROS concentration among AA women was approximately 9% higher than Caucasian women. MDA levels increased exponentially from non-disease control and precancerous tissues relative to malignant tissues. Furthermore, malignant serous ovarian samples showed significantly higher reactive carbonyl content compared to the non-disease controls (p=0.009), while GPX3 levels decreased considerably in serous cystadenoma and malignant tissue samples, and non-diseased control compared to borderline disease. The results suggest accumulation of ROS and MDA levels may be a causative factor for SOC. Elevated levels of MDA and reactive carbonyl proteins could override the GPX3 enzyme capacity therefore, initiating serous ovarian neoplasm.

Differential Measurements of Oxidatively Modified Proteins in Colorectal Adenopolyps.

INTRODUCTION: Adenopolyps patients have a three-fold higher risk of colon cancer over the general population, which increases to six-fold if the polyps are multiple and with lower survival among African American population. Currently, 6% of CRC can be ascribed to mutations in particular genes. Moreover, the optimal management of patients with colorectal adenopolyps depends on the accuracy of appropriate staging strategies because patients with similar colorectal adenocarcinoma architecture display heterogeneity in the course and outcome of the disease. Oxidative stress, due to an imbalance between reactive oxygen species (ROS) and antioxidant capacities as well as a disruption of redox signaling, causes a wide range of damage to DNA, proteins, and lipids which promote tumor formation. OBJECTIVE/METHOD: This study applied spectrophotometric, dinitrophenylhydrazone (DNPH) assay, two-dimensional gel electrophoresis, and western blot analyses to assess the levels of oxidatively modified proteins in 41 pairs of primary colorectal tissues including normal/surrounding, adenopolyps (tubular, tubulovillous, villous, polypvillous) and carcinoma. Analysis of variance (ANOVA) and Student's t-tests were utilized for the resulting data set. RESULTS: Our data showed that the levels of reactive protein carbonyl groups significantly increased as colorectal adenopolyps progresses to malignancy. No significant differences were found in the levels of carbonyl proteins between gender samples analyzed. For African American patients, there were, relative to Caucasians, 10% higher levels of reactive carbonyls in proteins of tubulovillous tissue samples (P < 0.05) and over 36% higher in levels in adenocarcinomas (P < 0.05). In normal tissues and tubular, there were no significant differences between the two groups in levels of protein carbonyls. Differences in the levels of protein carbonyl expression within individual patient samples with different number of tumor cells were notably evident. CONCLUSION: Results suggested that oxidative stress could be involved in the modification of oxidatively carbonyl proteins in the precancer stages, leading to increased aggressiveness of colorectal polyps.

Oxidatively modified proteins in the serous subtype of ovarian carcinoma.

Serous subtype of ovarian cancer is considered to originate from fallopian epithelium mucosa that has been exposed to physiological changes resulting from ovulation. Ovulation influences an increased in inflammation of epithelial ovarian cells as results of constant exposure of cells to ROS. The imbalance between ROS and antioxidant capacities, as well as a disruption of redox signaling, causes a wide range of damage to DNA, proteins, and lipids. This study applied spectrophotometric, dinitrophenylhydrazone (DNPH) assay, two-dimensional gel electrophoresis, and Western blot analyses to assess the levels of oxidatively modified proteins in 100 primary serous epithelial ovarian carcinoma and normal/surrounding tissues. These samples were obtained from 56 Caucasian and 44 African-American patients within the age range of 61 ± 10 years. Analyses showed that the levels of reactive protein carbonyl groups increased as stages progressed to malignancy. Additionally, the levels of protein carbonyls in serous ovarian carcinoma among African Americans are 40% (P < 0.05) higher relative to Caucasian at similar advanced stages. Results suggest that oxidative stress is involved in the modification of carbonyl protein groups, leading to increased aggressiveness of epithelial ovarian tumors and may contribute to the disease's invasiveness among African Americans.

Frequencies of mtDNA mutations in primary tissues of colorectal adenopolyps.

To investigate the potential role of mtDNA alterations during the onset of colorectal cancer, the occurrence of mtDNA variants in colorectal adenomatous (Tubular, Tubulovillous, and Villous) polyps, were studied. High resolution endonucleases and PCR-based sequence were applied to examine mtDNA variants in the ND and ATPase genes of 64 primary tissues of colorectal adeno-polyps and their matched normal controls. Forty-two variants were observed and 57% (24/42) were not previously reported in the MITODAT reference. Fifty-eight percent of these variants were germline and homoplasmic transitions. The distribution of observed mtDNA variants includes: 31% (13/42) tubular, 52% (22/42) tubulovillous, 45% (19/42) villous, and 45% (19/42) cancer (including FAP and JVP). Notably, an unreported germline variant in the ATPase 8 gene at nucleotide position (np) G8573A was observed in tubulovillous adenomas tissues. The results suggest that some specific mtDNA variants may serve as a potential biomarker for colorectal adenomatous polyps.

Analysis of mtDNA sequence variants in colorectal adenomatous polyps.

Colorectal tumors mostly arise from sporadic adenomatous polyps. Polyps are defined as a mass of cells that protrudes into the lumen of the colon. Adenomatous polyps are benign neoplasms that, by definition display some characteristics of dysplasia. It has been shown that polyps were benign tumors which may undergo malignant transformation. Adenomatous polyps have been classified into three histologic types; tubular, tubulovillous, and villous with increasing malignant potential. The ability to differentially diagnose these colorectal adenomatous polyps is important for therapeutic intervention. To date, little efforts have been directed to identifying genetic changes involved in adenomatous polyps. This study was designed to examine the relevance of mitochondrial genome alterations in the three adenomatous polyps. Using high resolution restriction endonucleases and PCR-based sequencing, fifty-seven primary fresh frozen tissues of adenomatous polyps (37 tumors and 20 matched surrounding normal tissues) obtained from the southern regional Cooperative Human Tissue Network (CHTN) and Grady Memorial Hospital at Atlanta were screened with three mtDNA regional primer pairs that spanned 5.9 kbp. Results from our data analyses revealed the presence of forty-four variants in some of these mitochondrial genes that the primers spanned; COX I, II, III, ATP 6, 8, CYT b, ND 5, 6 and tRNAs. Based on the MITODAT database as a sequence reference, 25 of the 44 (57%) variants observed were unreported. Notably, a heteroplasmic variant C8515G/T in the MT-ATP 8 gene and a germline variant 8327delA in the tRNAlys was observed in all the tissue samples of the three adenomatous polyps in comparison to the referenced database sequence. A germline variant G9055A in the MT-ATP 6 gene had a frequency of 100% (17/17) in tubular and 57% (13/23) in villous adenomas; no corresponding variant was in tubulovillous adenomas. Furthermore, A9006G variant at MT-ATP 6 gene was observed at frequency of 57% (13/23) in villous adenomas only. Interestingly, variants A9006G and G9055A were absent in the villous tissue samples that were clinicopathological designated as "polyvillous adenomas". Our current data provide a basis for continued investigation of certain mtDNA variants as predictors of the three adenomatous polyps in a larger number of clinicopathological specimens.

Synthesis and characterization of silver-nanoparticle-impregnated fiberglass and utility in water disinfection.

A number of researchers have deployed silver (Ag) nanoparticles through a number of techniques on various substrates including carbon, zeolites and polymers for water disinfection applications. However, Ag impregnated on an inorganic fiberglass surface through a simple electroless process was only recently reported for the first time. Fiberglass impregnated with Ag nanoparticles displays superior performance over carbon-based silver support systems but little is known about the factors that affect the architecture of the system, its interfacial properties and its consequent bactericidal activity. In this study, Ag content and particle size on a fiberglass substrate were manipulated by adjusting the AgNO(3) concentration, immersion time, temperature, solution pH and reduction temperature. The reduction chemistry of the Ag-nanoparticle-impregnated fiberglass is described and supported with thermal gravimetric analysis (TGA) and photoelectron spectroscopy (XPS) measurements. The Ag content along with the particle size and particle size distribution were characterized with scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS) and x-ray diffraction (XRD). The Ag content on the fiberglass mats ranged from 0.04 to 4.7 wt% Ag/g-fiber with a size distribution of 10-900 nm under standard processing conditions. Inductively coupled plasma mass spectrometry (ICP-MS) was used to analyze the Ag desorption from the fiberglass substrate, while the bactericidal properties were evaluated against Escherichia coli (E. coli).

Bactericidal activity of Ag nanoparticle-impregnated fibreglass for water disinfection.

A new bactericidal system composed of fibreglass impregnated with silver (Ag) nanoparticles was developed and tested. Silver content, particle size and distribution were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The antibacterial effectiveness was evaluated against Escherichia coli (E. coli, ATCC 29055). The minimum inhibitory loading was determined to be less than 1.8 wt% of silver nanoparticles per gram of fibreglass. In a 1 h immersion test, using a 0.1 mg fibreglass mat ml(-1), with 2.9 wt% loading of silver nanoparticles completely disinfected 100 ml of 10(6) CFU ml(-1) of E. coli, dramatically outperforming activated carbon fibres impregnated with silver. Inactivation rate studies of 0.05 mg fibreglass mat ml(-1) (Ag 1.8 wt%) with 10(12) CFU E. coli displayed a 7 log reduction in 5 minutes. The activation and reuse of fibreglass (Ag 4.3 wt%) maintained its full effectiveness after two cycles of use and thermal regeneration at 350 degrees C.

mtDNA sequence variants in subtypes of epithelial ovarian cancer stages in relation to ethnic and age difference.

Epithelial ovarian cancer is the fifth leading cause of cancer mortality among women in the United States. For this disease, differences in age-adjusted incidence and survival rates between African American and Caucasian women are substantial. The objective of this study was to examine mtDNA sequence variants in 118 frozen tissues of three subtypes of epithelial ovarian cancer (serous, n = 48 endometrioid, n = 47 and mucinous, n = 23) and matched paracancerous normal tissues (n = 18) in relation to racial/ethnic and age differences. Restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR)-based sequencing were used to evaluate two regions of mtDNA spanning 5317 to 7608 and 8282 to 10110 bp and including ND subunits 2, 3, MT-COI, II, and III, ATPase 8, a part of ATPase 6, and tRNA genes in frozen ovarian tissues obtained from the southern regional Cooperative Human Tissue Network (CHTN) and University of Alabama-Birmingham (UAB) Ovarian Spore Center. Thirty-nine mtDNA variants were detected of which 28 were previously unreported. One somatic variant of C9500T was observed. A variant, C7028T in the MT-CO1 gene, had an ascending frequency from borderline (8%) to stages III/IV (75%) among the three ovarian cancer subtypes and stages. It was found in 86% (42/49) of African-American and 43% (37/87) of the Caucasian women. A variant, T8548G in the ATPase 6 gene was detected at a frequency of 72% (18/25) in ovarian serous subtype tissues in stages III/IV. Of the African American patients under age 40, 95% (20/21) harbored the T8548G variant; this was in contrast to only 22% (8/35) of Caucasian patients in same age group. Variants C7256T and G7520A had a frequency of 54% (6/11) in endometrioid stage III; no corresponding variants were observed in mucinous subtype stage III. Furthermore, variants C7256T and G7520A were absent in serous ovarian cancer subtype. Interestingly, the C7520T variant in tRNA gene was present in 74% (36/49) of African American and 26% (23/87) of Caucasian patients. Taken together, our results suggest that, with respect to ethnic and age difference, these mtDNA variants may be involved in epithelial ovarian carcinogenesis.

Mitochondrial DNA sequence variants in epithelial ovarian tumor subtypes and stages.

BACKGROUND: A majority of primary ovarian neoplasms arise from cell surface epithelium of the ovaries. Although old age and a positive family history are associated risk factors, the etiology of the epithelial ovarian tumors is not completely understood. Additionally, knowledge of factors involved in the histogenesis of the various subtypes of this tumor as well as those factors that promote progression to advanced stages of ovarian malignancy are largely unknown. Current evidence suggests that mitochondrial alterations involved in cellular signaling pathways may be associated with tumorigenesis. METHODS: In this study, we determined the presence of polymorphisms and other sequence variants of mitochondrial DNA (mtDNA) in 102 epithelial ovarian tumors including 10 matched normal tissues that paired with some of the tumors. High-resolution restriction endonucleases and PCR-based sequencing were used to assess the mtDNA variants spanning 3.3 kb fragment that comprised the D-Loop and 12S rRNA-tRNAphe, tRNAval, tRNAser, tRNAasp, tRNAlys, ATPase 6, ATPase 8, cytochrome oxidase I and II genes. RESULTS: Three hundred and fifty-two (352) mtDNA sequence variants were identified, of which 238 of 352 (68%) have not been previously reported. There were relatively high frequencies of three mutations in the 12S rRNA gene at np 772, 773, and 780 in stage IIIC endometrioid tumors, two of which are novel (773delT and 780delC), and occurred with a frequency of 100% (7/7). Furthermore, two mutations were observed in serous tumors only at np 1657 in stage IV (10/10), and at np 8221delA in benign cystadenomas (3/3) and borderline tumors (4/4). A high frequency, 81% (13/16) of TC insertion at np 310 was found only in early stages of serous subtype (benign cystadenomas, 3/3; borderline tumors, 4/4; stage I tumors, 2/5 and matched normal tissues 4/4). CONCLUSION: Our findings indicate that certain mtDNA mutations can reliably distinguish the different histologic subtypes of epithelial ovarian tumors. In addition, these data raise the possibility that certain mtDNA mutations may be useful biomarkers for predicting tumor aggressiveness and may play a potential role in tumorigenesis.

Efficacy of oligodynamic metals in the control of bacteria growth in humidifier water tanks and mist droplets.

Antimicrobial capsules were evaluated for their effectiveness to control bacterial contamination of cool mist humidifiers. These capsules contain a mixture of silver and copper promoted alumina beads designed to release low concentrations of these oligodynamic metals into the reservoir water for bacteria control. The reservoir water and mist droplets from the humidifier units were tested for the presence of bacteria over a three-week period. A control unit (without capsule) showed significant bacterial contamination by day three, which increased throughout the three-week test period, in both the reservoir and mist droplets, whereas the antimicrobial capsules reduced contamination during the first week, and minimized the presence of bacteria, in both the reservoir water and mist droplets, to less than 2% of the control unit throughout the three-week test period. It was also observed that, after each inactive weekend, the initial discharge of bacteria via the mist droplets in the control unit was significantly higher than during daily use. However, initial bacterial discharge from the test unit following weekend inactivity never exceeded 0.5% of the control unit. In conclusion, these capsules containing oligodynamic metals are effective in controlling bacteria growth in humidifier water tanks and mist droplets.