Does the environment affect menopause? A review of the effects of endocrine disrupting chemicals on menopause.

Endocrine disrupting chemicals are widely distributed in our environment. Humans are exposed to these compounds not only through their occupations, but also through dietary consumption and exposure to contaminated water, personal care products and textiles. Chemicals that are persistent in the body and in our environment include dioxins and polychlorinated biphenyls. Non-persistent chemicals including bisphenol A, phthalates and parabens are equally as important because they are ubiquitous in our environment. Heavy metals, including lead and cadmium, can also have endocrine disrupting properties. Although difficult to study due to their variety of sources of exposures and mechanisms of action, these chemicals have been associated with early menopause, increased frequency of vasomotor symptoms, altered steroid hormone levels and markers of diminished ovarian reserve. Understanding the impacts of these exposures is important given the potential for epigenetic modification, which can alter gene function and result in multi-generational effects. This review summarizes findings in humans and animals or cell-based models from the past decade of research. Continued research is needed to assess the effects of mixtures of chemicals, chronic exposures and new compounds that are continuously being developed as replacements for toxic chemicals that are being phased out.

Intratumoral delivery of tavokinogene telseplasmid yields systemic immune responses in metastatic melanoma patients.

BACKGROUND: Interleukin 12 (IL-12) is a pivotal regulator of innate and adaptive immunity. We conducted a prospective open-label, phase II clinical trial of electroporated plasmid IL-12 in advanced melanoma patients (NCT01502293). PATIENTS AND METHODS: Patients with stage III/IV melanoma were treated intratumorally with plasmid encoding IL-12 (tavokinogene telseplasmid; tavo), 0.5 mg/ml followed by electroporation (six pulses, 1500 V/cm) on days 1, 5, and 8 every 90 days in the main study and additional patients were treated in two alternative schedule exploration cohorts. Correlative analyses for programmed death-ligand 1 (PD-L1), flow cytometry to assess changes in immune cell subsets, and analysis of immune-related gene expression were carried out on pre- and post-treatment samples from study patients, as well as from additional patients treated during exploration of additional dosing schedules beyond the pre-specified protocol dosing schedule. Response was measured by study-specific criteria to maximize detection of latent and potentially transient immune responses in patients with multiple skin lesions and toxicities were graded by the Common Terminology Criteria for Adverse Events version 4.0 (CTCAE v4.0). RESULTS: The objective overall response rate was 35.7% in the main study (29.8% in all cohorts), with a complete response rate of 17.9% (10.6% in all cohorts). The median progression-free survival in the main study was 3.7 months while the median overall survival was not reached at a median follow up of 29.7 months. A total of 46% of patients in all cohorts with uninjected lesions experienced regression of at least one of these lesions and 25% had a net regression of all untreated lesions. Transcriptomic and immunohistochemistry analysis showed that immune activation and co-stimulatory transcripts were up-regulated but there was also increased adaptive immune resistance. CONCLUSIONS: Intratumoral Tavo was well tolerated and led to systemic immune responses in advanced melanoma patients. While tumor regression and increased immune infiltration were observed in treated as well as untreated/distal lesions, adaptive immune resistance limited the response.

Designing a Uniaxial Tension/Compression Test for Springback Analysis in High-Strength Steel Sheets.

We describe an innovative design for an in-plane measurement technique that subjects thin sheet metal specimens to bidirectional loading. The goal of this measurement is to provide the critical performance data necessary to validate complex predictions of the work hardening behavior during reversed uniaxial deformation. In this approach, all of the principal forces applied to the specimen are continually measured in real-time throughout the test. This includes the lateral forces that are required to prevent out of plane displacements in the specimen that promote buckling. This additional information will, in turn, improve the accuracy of the compensation for the friction generated between the anti-bucking guides and the specimen during compression. The results from an initial series of experiments not only demonstrate that our approach is feasible, but that it generates data with the accuracy necessary to quantify the directionally-dependent changes in the yield behavior that occur when the strain path is reversed (i.e., the Bauschinger Effect).

The Influence of Post-Build Microstructure on the Electrochemical Behavior of Additively Manufactured 17-4 PH Stainless Steel.

The additive manufacturing (AM) build process produces a segregated microstructure with significant variations in composition and phases that are uncommon in traditional wrought materials. As such, the relationship between the post-build microstructure and the corrosion resistance is not well understood. Stainless steel alloy 17-4PH is an industrially-relevant alloy for applications requiring high-strength and good corrosion resistance. A series of potentiodynamic scans conducted in a deaerated 0.5 mol/L NaCl solution evaluated the influence of these microstructural differences on the pitting behavior of SS17-4. The pitting potentials were found to be higher in the samples of additively-processed material than in samples of the alloy in wrought form. This indicates that the additively-processed material is more resistant to localized corrosion and pitting in this environment than the wrought alloy. The results also suggest that after homogenization, the additively-produced SS17-4 could be more resistant to pitting than wrought SS17-4 in an actual service environment.

Carbonic anhydrase IX (CA-IX) and high-risk human papillomavirus (H-HPV) as diagnostic biomarkers of cervical dysplasia/neoplasia in Japanese women with a cytologic diagnosis of atypical glandular cells (AGC): a Gynecologic Oncology Group (GOG) Study.

BACKGROUND: High-risk human papillomavirus (H-HPV) infection is linked to cervical neoplasia but its role in detecting cervical glandular lesions (GLs) is unclear. Carbonic anhydrase IX (CA-IX) is a hypoxic biomarker that is highly expressed in neoplastic cervical GLs. The diagnostic utility of these biomarkers was evaluated by the Gynecologic Oncology Group in Japanese women with a cytological diagnosis of atypical glandular cells. METHODS: Immunostaining was used to detect CA-IX in a conventional Pap smear. Immunoreactivity of CA-IX was interpreted by a panel of pathologists blinded to the histological diagnosis. Polymerase chain reaction was used to detect H-HPV in a liquid-based cytology specimen. RESULTS: Significant cervical lesions (SCLs), defined as cervical intraepithelial neoplasia (CIN2, CIN3), adenocarcinoma in situ or invasive carcinoma, were observed in 37/88 (42%) of women. CA-IX testing alone (n=88) had a sensitivity of 89, 100 or 73% for SCLs, GLs or significant squamous lesions (SLs), respectively, with a false negative rate (FNR) of 14%. Testing for H-HPV (n=84) had a sensitivity of 65, 53 or 80% for SCLs, GLs or SLs, respectively, with a FNR of 22%. The combination of CA-IX and H-HPV testing had a sensitivity of 97, 100 or 93% for SCLs, GLs or SLs, respectively, with a FNR of 5%. Among eight H-HPV-negative GLs, six (75%) had a diagnosis of lobular endocervical glandular hyperplasia (LEGH). CONCLUSION: The combination of CA-IX and HPV testing improved the diagnostic accuracy. The low rate of H-HPV positivity in the GLs was associated with coexisting LEGH independent of H-HPV.

Electron transport in gold nanowires: stable 1-, 2- and 3-dimensional atomic structures and noninteger conduction states.

Experimental conductivity measurements made during highly stable tensile deformation of Au nanowires show a rich variety of behaviors, including noninteger quantum conductance plateaus, transitions, and slopes. Using tight binding conductance calculations on simulated nanowires previously deformed using density functional theory, we demonstrate that all of these phenomena arise from structural transitions between deeply metastable ordered atomic configurations that self-organize during tensile deformation.

Acute phase reactants ceruloplasmin and haptoglobin and their relationship to plasma prostaglandins in rabbits bearing the VS2 carcinoma.

Results of previous studies have shown that the VX2 carcinoma in rabbits synthesizes large amounts of prostaglandin E2 (PGE2). PGE2 secreted by the tumor is rapidly metabolized and can be measured in plasma as the metabolite 13,14-dihydro-15-keto-PGE2 (PGE2-M). We have previously proposed that the hypercalcemia that occurs in rabbits bearing the VX2 carcinoma is due to excessive secretion of PGE2 by the tumor and its subsequent action on the skeleton as a bone resorption-stimulating factor. In the course of these studies, we noted that the plasma of rabbits bearing the VS2 carcinoma became blue about 1 wk after tumor implantation. The intensity of the color increased markedly thereafter. We therefore measured ceruloplasmin in plasma by both chemical and immunological assay methods. Plasma ceruloplasmin and PGE2-M rose in parallel (within 7-10 days) and preceded by 7-10 days the development of hypercalcemia. 2 wk after tumor implantation, plasma PGE2-M and ceruloplasmin had risen about 20- and 6-fold, respectively, while the rise in plasma calcium was just beginning. Indomethacin, an inhibitor of prostaglandin synthesis, given from the time of tumor implantation prevented completely the hypercalcemia and largely inhibited the rise in ceruloplasmin. When given after hyperprostaglandinemia had developed, indomethacin produced a fall in both PGE2-M and ceruloplasmin. A rise in plasma haptoglobin concentrations similar to that seen for ceruloplasmin was also observed. No changes in plasma albumin concentrations occurred. We conclude that the acute phase reactants ceruloplasmin and haptoglobin rise rapidly in the plasma of rabbits bearing the VX2 carcinoma, and that this increase is related to arachidonic acid metabolism in these animals. It is possible that arachidonic acid metabolites also play a role in the elevations of these two plasma proteins observed in certain patients with malignant tumors.

Evidence that the bone resorption-stimulating factor produced by mouse fibrosarcoma cells is prostaglandin E 2 . A new model for the hypercalcemia of cancer.

A transplantable mouse fibrosarcoma, HSDM(1), produces a potent bone resorption-stimulating factor. The factor can be extracted from the tumor tissue and harvested from the medium of clonal strains of HSDM(1) tumor cells growing in monolayer culture. It has several chemical and biological properties of a prostaglandin. Using radioimmunoassay techniques, we have shown that HSDM(1) cells synthesize and secrete large quantities of prostaglandin E(2) (PGE(2)). The specific bone resorption-stimulating activity of the HSDM(1) factor extracted from the tumor is high and approximately equal to that of PGE(2) as measured in a bone tissue culture system in vitro. Indomethacin, a potent inhibitor of PGE(2) synthesis in HSDM(1) cells, also inhibits production by the cells of the bone resorption-stimulating factor, and has no detectable nonspecific effects on the bone culture assay system. Mice bearing the HSDM(1) tumor have higher levels of both calcium and PGE(2) in serum than control mice. We conclude that PGE(2) is the bone resorption-stimulating factor produced by HSDM(1) tumor cells, and that secretion of PGE(2) by the tumor in vivo accounts for the relative hypercalcemia observed in tumor-bearing animals. The HSDM(1) tumor cell system constitutes a new model for studying the pathogenesis of hypercalcemia associated with certain malignant tumors.

The serologic specificity of tropocollagen telopeptides.

Tropocollagen preparations from carp, buffalo fish, rats, calves, sheep, and humans have been studied by electron microscopy and serologic methods. Tropocollagens from each species appeared identical by electron microscopy but they were readily distinguished (except between sheep and calves) by C'-fixation tests with rabbit antisera against the various tropocollagens. Tests with calf tropocollagen antiserum showed no distinction between tropocollagen isolated from different tissues nor between individuals of the same or different strains. The major immunogenic sites in native tropocollagen are the telopeptides, and these are present on both alpha1- and alpha2-chains. The C'-fixing activity was lost with heat denaturation of the tropocollagen, but could be recovered in a concentration-dependent process on cooling. The fact that pure and enzyme-treated collagen can provoke serologic reaction implies that collagenous sutures and prostheses used in surgery may lead to sensitization and rejection, a fact which may merit clinical concern.

Mouse sperm basic nuclear protein. Electrophoretic characterization and fate after fertilization.

Mouse sperm were labeled in vivo with [3H]arginine. The sperm were then followed autoradiographically from the time of label incorporation until after fertilization. The label was completely lost from the sperm head after fertilization, during the oocyte's second meiotic division. That the [3H]arginine was incorporated into a sperm-specific basic protein was demonstrated by fractionating acid extracts of epididymal and ejaculated sperm with polyacrylamide gel electrophoresis. All the histone fractions were resolved in the epididymal extracts, but in addition a band was present that migrated faster than histone F2al and slower than the salmon protamine used as a marker. This new fraction (proposed name: musculine) was also present in ejaculated sperm; it was shown to be the only fraction that was labeled. Musculine therefore represents the end product of a histone transition in mice. It is, however, according to our electrophoretic characterization, not identical to the classical fish protamines. Rather, musculine resembles bovine sperm nuclear protein. Since the loss of this fraction from the sperm head was coincident with the rearrangement of the male genome, before its resumption of transcription, it is suggested that musculine is involved in the control of chromatin that accompanies spermiogenesis and fertilization.

Establishment of a clonal strain of hepatoma cells which secrete albumin.

A clonal strain of epithelial cells (designated MH(1)C(1)) has been established from the transplantable Morris hepatoma No. 7795. The cells have maintained distinctive morphology throughout more than 20 subcultures (split 1:5) at 2- to 4-week intervals in supplemented Ham's F 10 medium. They contain many highly refractile, round, cytoplasmic bodies which stain bright red with Oil Red O. The population doubling time was 2 wk when the clonal strain was first established. It has gradually decreased to 1 wk. The cells synthesize rat serum albumin and secrete it into the culture medium as determined immunologically by microcomplement fixation and double diffusion. Albumin secretion (3-6 microg albumin/mg cell nitrogen/24 hr) occurs throughout the logarithmic phase of cell proliferation and has not diminished during serial propagation since the strain was initiated 15 months ago.

Control of growth hormone production by a clonal strain of rat pituitary cells. Stimulation by hydrocortisone.

Addition of hydrocortisone to the medium of a clonal strain of rat pituitary cells (GH(3)) stimulated the rate of production of growth hormone. The stimulation had a lag period of about 24 hr, reached a maximum at 70-100 hr, and was observed at a hydrocortisone concentration as low as 5 x 10(-8)M. Cells maximally stimulated with 3 x 10(-6)M hydrocortisone produced 50-160 microg growth hormone/mg cell protein/24 hr. These rates were four to eight times those observed in control cells. At maximum stimulation, intracellular levels of growth hormone in both stimulated and control cells were equal to the amount secreted into the medium in about 15 min. Removal of hydrocortisone from the medium of GH(3) cells caused a return of the rate of growth hormone production to that in control cells. Addition of hydrocortisone to the medium of cells growing exponentially with a population-doubling time of 60 hr caused both an increase in the doubling time to 90 hr and a stimulation of growth hormone production. Cycloheximide (3.6 x 10(-5)M) and puromycin (3.7 x 10(-4)M) suppressed incorporation of labeled amino acids into protein by 93 and 98%, respectively, and suppressed growth hormone production by stimulated and control cells by at least 94%.

Production of both prolactin and growth hormone by clonal strains of rat pituitary tumor cells. Differential effects of hydrocortisone and tissue extracts.

Several established clonal strains of rat pituitary cells which produce growth hormone in culture have been shown to secrete a second protein hormone, prolactin. Prolactin was measured immunologically in culture medium and within cells by complement fixation. Rates of prolactin production varied from 6.6 to 12 microg/mg cell protein per 24 hr in four different cell strains. In these cultures ratios of production of prolactin to growth hormone varied from 1.0 to 4.1. A fifth clonal strain produced growth hormone but no detectable prolactin. Intracellular prolactin was equivalent to the amount secreted into medium in a period of about 1-2 hr. Both cycloheximide and puromycin suppressed prolactin production by at least 94%. Hydrocortisone (3 x 10(-6)M), which stimulated the production of growth hormone 4- to 8-fold in most of the cell strains, reduced the rate of prolactin production to less than 25% of that in control cultures. Conversely, addition of simple acid extracts of several tissues, including hypothalamus, to the medium of all strains increased the rate of production of prolactin six to nine times and decreased growth hormone production by about 50%. We conclude that multifunctional rat pituitary cells in culture show unusual promise for further studies of the control of expression of organ-specific activities in mammalian cells.

Differentiated rat glial cell strain in tissue culture.

Rat glial tumors, induced by injections of N-nitrosomethylurea, were plated and propagated in culture. Among a few cell strains obtained, one clone contains S-100 protein, which is unique to brain in vertebrates. Stationary-phase cultures contain approximately ten times more S-100 protein per cell than exponentially growing cells. When injected into newborn rats, cells producing S-100 grew as a glial tumor, which contained S-100 protein.

Antibodies to photoproducts of deoxyribonucleic acids irradiated with ultraviolet light.

A rabbit immunized with complexes of methylated bovine serum albumin and ultraviolet-irradiated DNA from calf thymus produced antibodies directed toward the photoproducts in the DNA. Serologic activity appeared after irradiation of DNA at 270 mmicro and decreased upon irradiation at 235 mmicro. The antigenic determinants of the ultraviolet-treated DNA appear to be photoproducts associated primarily with thymine, as measured by direct dependence of serologic activity on the adenine-thymine content of the DNA, and by inhibition of the Serlolgic reaction by the irradiated di-,tri-,and tetra-(thymidine-5'-phosphate nucleotides.

Synthesis of compounds with properties of leukotrienes C4 and D4 in gerbil brains after ischemia and reperfusion.

6-Sulfidopeptide-containing leukotriene-like immunoreactivity was synthesized in gerbil forebrains after bilateral common carotid occlusion and reperfusion. At 5, 10, or 15 minutes of ischemia, concentrations increased significantly and became more marked on reperfusion. Immunoreactivity was highest in forebrain gray matter and was below the detection limit of the assay in brain regions remote from the zone of ischemia. In vitro experiments with vascular cells and organ cultures of cerebral arteries indicate that the cerebral blood vessel wall is not a major source of biosynthetic activity in the brain. These experiments demonstrate leukotriene biosynthesis by the brain. Because synthesis occurs during ischemia and reperfusion and because leukotrienes are potent vasoconstrictors and promoters of tissue edema, they may play a role in the pathophysiology of cerebral ischemia.

Topographic Measurement of Individual Laser Tracks in Alloy 625 Bare Plates.

Additive manufacturing (AM) combines all of the complexities of materials processing and manufacturing into a single process. The digital revolution made this combination possible, but the commercial viability of these technologies for critical parts may depend on digital process simulations to guide process development, product design, and part qualification. For laser powder bed fusion (LPBF), one must be able to model the behavior of a melt pool produced by a laser moving at a constant velocity over a smooth bare metal surface before taking on the additional complexities of this process. To provide data on this behavior for model evaluations, samples of a single-phase nickel-based alloy were polished smooth and exposed to a laser beam at 3 different power and speed settings in the National Institute of Standards and Technology (NIST) Additive Manufacturing Metrology Testbed (AMMT) and a commercial AM machine. The solidified track remaining in the metal surface after the passing of the laser is a physical record of the position of the air-liquid-solid interface of the melt pool trailing behind the laser. The surface topography of these tracks was measured and quantified using confocal laser scanning microscopy (CLSM) for use as benchmarks in AM model development and validation. These measurements are part of the Additive Manufacturing Benchmark Test Series (AM-Bench).