Search

Advances in high-throughput sequencing (HTS) technologies have greatly increased the availability of genomic data and potential discovery of clinically significant genomic variants. However, numerous issues still exist with the analysis of these data, including data complexity, the absence of formally agreed upon best practices, and inconsistent reproducibility. Toward a more robust and reproducible variant-calling paradigm, we propose a series of selective noise filtrations and post-alignment quality control (QC) techniques that may reduce the rate of false variant calls. We have implemented both novel and refined post-alignment QC mechanisms to augment existing pre-alignment QC measures. These techniques can be used independently or in combination to identify and correct issues caused during data generation or early analysis stages. The adoption of these procedures by the broader scientific community is expected to improve the identification of clinically significant variants both in terms of computational efficiency and in the confidence of the results., Availability: https://hive.biochemistry.gwu.edu/., (Copyright © 2017. Published by Elsevier Inc.)

Among the retroposons, the source of the endonuclease activity is known to be variable and can be provided as either a retroviral-like integrase or a protein similar to the cellular apurinic-apyrimidinic endonuclease. It has also been reported that other retroposon and retrointron sequences have limited similarity to various eubacterial endonucleases. We investigated whether any retroposon genomes possibly encode multiple endonuclease functions. Amino acid alignments were generated and analyzed for the presence of the characterized ordered-series-of-motifs (OSM) representative of four different endonuclease functions. The results indicate that SLACS, CZAR, CRE1, CRE2, and some Trypanosoma brucei retroposon sequences encode multiple putative endonuclease functions. Interestingly, one of the endonuclease functions is embedded within the potential ribonuclease H sequence found in SLACS, CZAR, CRE1, CRE2, and R2BM retroposons.

The interaction of cells with their substratum is an important determinant of cell behaviour, influencing attachment, proliferation, and motility. Such interactions are mediated by cell surface receptors which bind to attachment factors, like the glycoprotein laminin in basement membranes. We have previously shown that expression of the 67-kDa laminin receptor (67LR) is elevated in proliferating retinal microvasculature compared with mature, quiescent vessels. Here, we examined 67LR mRNA and protein expression in primary cultures of retinal microvascular endothelial cells (RMEC) and in the breast cancer cell-line T47D during stages of contact inhibition. In both cell types, the expression levels of 67LR mRNA and membrane-associated 67LR protein were significantly increased during the proliferative phases of monolayer formation. As the cells achieved contact inhibition, 67LR expression was reduced to comparatively low levels. Thus, the differential expression of 67LR between dividing and contact-inhibited cells may indicate a role for this receptor during proliferative processes., (Copyright 2000 Academic Press.)

Plasma levels of 17beta-estradiol, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-P), and testosterone were measured in adult female coho salmon in late vitellogenesis, approximately 1.5 months before spawning and just before and following intraperitoneal injection with the aromatase inhibitor (AI) Fadrozole. Injection at dosages of 0.1, 1.0, and 10.0 mg AI/kg body wt caused a significant drop in plasma 17beta-estradiol levels relative to preinjection values within 3 or 6 h. Injection of 10 mg AI/kg body wt caused a significant increase in plasma 17alpha-20beta-P levels within 3 h. Ten days after injection 67% of the fish treated with 10 mg AI/kg body wt had ovulated in contrast with 0% in the group injected with 0.1 mg AI/kg body wt. The fertilization rate of the eggs varied between 96% in the control group and 85% in the groups injected with AI. We conclude that the shift from 17beta-estradiol to 17alpha,20beta-P biosynthesis, which is characteristic of maturing Oncorhynchus sp., was advanced significantly by treatment with AI and that Fadrozole can be used as a tool to investigate periovulatory endocrine changes in salmon., (Copyright 1999 Academic Press.)

A variety of endogenous and exogenous factors can influence sex steroid production by salmon ovarian follicles and ultimately impact reproductive development. We examined the effect of an aromatase inhibitor, fadrozole, and common environmental contaminants (PAHs) on sex steroid secretion by ovarian follicles. Ovarian follicles of coho salmon were incubated in vitro with various concentrations of testosterone (0.10-0.40 microM) and fadrozole (10 and 100 microM), or with varying doses (between 0.05 and 5.0 microM) of the PAHs beta-naphthoflavone (BNF) and 20-methylcholanthrene (20-MC). 17 beta-Estradiol secretion was significantly reduced when follicles were incubated in the presence of fadrozole, BNF, or 20-MC. In contrast, 17 beta-estradiol production by ovarian follicles increased in a dose-dependent manner when incubated with increasing doses of the aromatizable androgen testosterone. Although increasing doses of PAHs significantly reduced follicular 17 beta-estradiol production no effect on testosterone secretion was observed. Hence, both fadrozole and PAHs can significantly reduce 17 beta-estradiol secretion by salmon ovarian follicles and may affect female sexual development.

Juvenile coho salmon (Oncorhynchus kisutch) were injected with one of two recombinant bovine hormones, growth hormone (bGH; 5.0 and 0.5 micrograms.g-1 body wt) or placental lactogen (bPL; 5.0, 0.5, and 0.05 micrograms.g-1 body wt) to determine the effect on growth, plasma cortisol concentration, cytosolic corticosteroid receptors (CR) in the gills, and the development of hypoosmoregulatory ability. One week following a single injection or six weekly injections of bGH or bPL, the fish were measured and sampled for CR concentration and Na+,K(+)-ATPase activity in the gills. Fish were also challenged with salt water (salinity 25%) for 24 hr to determine saltwater tolerance at the end of the 6-week treatment. Treatment with bPL and bGH significantly increased weight and length of the fish. The 0.05-micrograms bPL dose significantly elevated plasma cortisol concentration, whereas all other hormone treatments did not affect cortisol levels. bPL and bGH also significantly increased CR concentration and Na+,K(+)-ATPase activity in the gills. The perturbation in plasma sodium concentration was least in animals receiving the highest dose of bPL and the bGH-treated animals following transfer to seawater. An increase in cytosolic CR by bGH and bPL may increase responsiveness of the gills to cortisol and partially account for the increase in Na+,K(+)-ATPase activity and greater ability to regulate plasma sodium in seawater as exhibited by the experimental groups.

Juvenile coho salmon were treated with bovine placental lactogen (bPL) and bovine growth hormone (bGH) to examine the growth promoting activities of these proteins in a lower vertebrate. Fish were intraperitoneally injected either with 0.5 or 5.0 micrograms/g bPL or with 5.0 micrograms/g bGH once a week for 5 weeks. After only a single injection and 1 week of growth, the high dose of bPL stimulated a significant increase in weight and length relative to untreated fish or fish treated with a control protein, bovine serum albumin. At the end of the experiment, all hormone-treated groups were significantly larger than controls. Fish treated with 5 micrograms/g bPL gained more than three times as much weight as controls. The 5.0 micrograms/g bGH group grew at the same rate as fish treated with one-tenth this dose of bPL, indicating that bPL is a potent stimulator of growth in this species. Radioreceptor assays performed on coho salmon liver membrane preparations indicate that bPL binds with approximately 430-fold higher affinity than bGH, and some 8000-fold higher affinity than bovine prolactin. The action of bPL relative to the structure and function of salmonid pituitary hormones is discussed.

The relative potency of several androgens to induce the male phenotype in sexually undifferentiated genotypic female chinook salmon were compared in two separate experiments. The aromatizable and nonaromatizable androgens testosterone (T) and 11-ketotestosterone (11-KT), and the synthetic aromatizable and nonaromatizable androgens 17 alpha-methyltestosterone (MT) and 17 alpha-methyldihydrotestosterone (MDHT) were administered to newly hatched alevins in a single 2-hr immersion treatment at doses ranging from 3.2 micrograms/liter to 10 mg/liter. The influence of these treatments on sex differentiation was evaluated by the histological examination of the resulting gonads 6 and 11 months later. In the control group, which was not exposed to exogenous steroids, no males or intersex fish were observed. In contrast, essentially 100% masculinization occurred in groups exposed to MDHT at dosages of 400 micrograms/liter and higher. Treatment with the aromatizable androgen MT resulted in a dose-dependent masculinization, with the production of 100% males at 400 micrograms/liter. However, higher doses resulted in fewer males. 11-KT and T were less potent than the synthetic androgens. The number of males produced after treatment with 11-KT followed a dose-dependent pattern while T showed virtually no masculinizing effect in inducing male phenotype in these studies. The resultant AD50 dosage levels (dosage at which 50% of the genotypic females were sex-reversed into phenotypic males) after a single 2-hr immersion treatment were: 30, 60, and 500 micrograms/liter for MDHT, MT, and 11-KT, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Twenty-seven patients with operable Stage II cervical cancer with a mean diameter tumor of 8.0 cm were studied in a feasibility study using preoperative 252Cf implants plus whole-pelvis radiation to 45 Gy followed by extrafascial total abdominal hysterectomy and bilateral salpingo-oophorectomy 4 to 6 weeks later. Hysterectomy specimens were studied by a set protocol. With the protocol used, 13/27 (51%) specimens and abdominal stagings were negative for residual tumor. The survival rate for the patients with negative findings was 93% at 5 years. In 14/27 (49%) patients the specimens were positive for residual tumor. In contrast, the 5-year survival rate for this group was 46% (P less than 0.001). In these patients several interrelated factors were determined to be of importance, i.e., (1) tumor size greater than 8 cm in maximum diameter, (2) positive or negative residual tumor status, and (3) total dose of radiation given. Survival was lower for larger tumors and specimens were more likely to show residual tumor. A lower treatment dose led to more positive specimens, as well as to poorer survival. While the patients with Stage II disease fared very well when negative specimens were found, further prospective studies of the appropriate treatment for those with positive tumor specimens are needed.

The influence of estrogen (E) and antiestrogen (AES) on the in vitro growth of BG-1 ovarian carcinoma cells, which express steroid receptors was examined (K. R. Geisinger, T. E. Kute, M. J. Pettenati, C. E. Welander, Y. Dennard, L. A. Collins, and M. E. Berens, Characterization of a human ovarian carcinoma cell line with estrogen and progesterone receptors, Cancer 63, 280-288, 1989). All determinations were simultaneously referenced under similar conditions to MCF-7 cells, a well-established cell line for modeling hormonal responses in breast cancer. In "complete" media containing fetal calf serum (FCS, 10%), MCF-7 cell numbers increased approximately 7 x in 7 days, remaining at this level Days 8-15. In contrast, BG-1 cells achieved similar numbers by Day 7, but showed apparent exponential growth over Days 8-15 to 15-20 x. Phenol red-free media containing 10% FCS (less than 20 pg estradiol (E2)/ml by RIA) was used to assess responses to E and AES. Growth of both MCF-7 and BG-1 cells slowed in E-free media. E2 (10 nM) stimulated the growth of both cell lines, yet was responsible for exponential increases during Days 8-15 only in BG-1 cell numbers (50-70 x). The metabolically active AES (4OH-tamoxifen, 50 nM) reduced E2-stimulated MCF-7 growth to 3-4 x, while tamoxifen (50 nM) had no effect. Rescue with 10 microM E2 fully overcame the AES inhibition of MCF-7 proliferation. In contrast, BG-1 cells experienced significant E2-stimulated growth reductions in the presence of either 4OH-tamoxifen or tamoxifen. E2 was observed to rescue BG-1 cells from both of these antagonists. We conclude that BG-1 ovarian carcinoma cells respond in vitro to E and AES. Moreover, by virtue of responses to tamoxifen, BG-1 cells may have an intrinsic capacity to hydroxylate tamoxifen to its active metabolite. This property of ovarian carcinoma cells might be worth exploiting in the design of more effective combination chemotherapy regimens.

Karyotypic analysis of tumor specimens from 29 patients with untreated epithelial ovarian carcinoma was performed at the University of Kentucky Medical Center. Twenty-three of the twenty-nine tumors had adequate cells for analysis. Seventeen of these tumors exhibited chromosome abnormalities. Chromosome alterations were complex, with an average of seven different abnormal chromosomal patterns per tumor (range 2-14). Chromosomes 1 and 11 were the most commonly involved, being abnormal in 89 and 83% of tumors, respectively. Chromosomes 3 and 7 were also frequently abnormal. In contrast to invasive tumors, alterations in chromosomes 1 and 11 were not seen in the two tumors of borderline malignant potential. Evidence for DNA amplification of IGF2, Ha-ras-1, and c-ets was not observed. Amplification of the c-erbB-2 oncogene was present in two tumors. These findings indicate that multiple karyotypic abnormalities occur in untreated epithelial ovarian malignancies, with chromosomes 1 and 11 being the most frequently abnormal. These data also suggest that alterations of these chromosomes may be associated with the biologically aggressive behavior of frankly invasive ovarian tumors.

The efficacy of carbon dioxide laser therapy in the treatment of cervical intraepithelial neoplasia (CIN) was evaluated in 253 patients treated at the University of Kentucky Medical Center from 1984 to 1987. All patients had histologically confirmed CIN and were treated in an outpatient setting. Parameters examined included severity of neoplasia, presence of koilocytosis on biopsy, depth of laser ablation, and the number of cervical quadrants involved by CIN. Following therapy, patients were examined at 3-month intervals from 12 to 48 months (mean 18 months). Eighty-nine per cent of patients were successfully treated with one laser ablation and all patients were cured of disease with two treatments. Over 70% of patients initially failing therapy had CIN III or greater than or equal to 3 quadrant disease. The most significant predictor of failure was lesion size. Only 74% of women with 3 or 4 quadrant disease were successfully treated with one treatment. No patient experienced postoperative infection or bleeding requiring treatment. These data confirm that carbon dioxide laser therapy is a safe and highly effective treatment method for all forms of CIN.

Thirty-two patients with Stage III or IV epithelial ovarian cancer and residual tumor volumes in excess of 2 cm in diameter after initial debulking were treated with platinum and cyclophosphamide chemotherapy. Twenty-seven patients (84%) received at least six courses of chemotherapy. Six patients developed grade 3 or 4 hematologic toxicity and one patient died with granulocytopenia and sepsis. The actuarial survival of the total group of patients was 78% at 12 months, 27% at 24 months, and 11% at 36 month. Of patients with residual disease 2-4 cm in diameter, 82% were alive 12 months after diagnosis and 46% were alive at 24 months. In contrast, patients with greater than 4 cm residual disease had a 12-month survival of 73% and a 24-month survival of only 7%. The size of residual tumor after surgery remains a very important prognostic factor in patients treated with platinum-based combination chemotherapy. Reoperation with further tumor debulking should be considered in ovarian cancer patients referred for chemotherapy with large volume residual disease to maximize response to combination chemotherapy.

A radioimmunoassay (RIA) capable of determining blood ACTH levels in salmonid fishes was developed and validated. The RIA used an antibody raised against mammalian ACTH, iodinated human ACTH as tracer, and human 1-39 ACTH as standard. Incubation of the standard or unknown with antibody for 3 days before addition of as little high-specific activity tracer as practicable (1500 cpm; equivalent to 5 pg ACTH) produced a very sensitive RIA; the operating range was 5 to 200 pg ACTH/ml. Extracts of both pars distalis and neurointermediate lobe of the pituitary glands from a range of salmonid species diluted parallel to the ACTH standard in the RIA. There was always considerably more ACTH-immunoreactivity (ACTH-IR) in the pars distalis extracts than in the neurointermediate lobe. Generally plasmas also diluted parallel to the ACTH standard, with the exception only of the plasma from sexually mature female salmonids, which diluted very non-parallel to the standard, leading to unrealistically low estimates of the ACTH-IR level. The use of heparin as an anticoagulant during collection of samples caused problems when these plasmas were immunoassayed; instead EDTA was found to be a suitable anticoagulant. When the ACTH-IR was extracted from a pool of plasma obtained from acutely stressed salmon and chromatographed on a column of BioGel P6, followed by subsequent ACTH RIA of the fractions, only a single sharp peak of ACTH-IR was detected, which eluted in the position of authentic 1-39 ACTH. The plasma ACTH-IR level in unstressed fish was low, and near the detection limit of the RIA. An acute stress, produced by crowding and confinement for 30 min, increased ACTH-IR approximately 10-fold, and plasma cortisol levels 50-fold, but the plasma alpha-MSH level was not affected. Dexamethasone-treated fish did not respond to this stressor with any increase in either ACTH or cortisol levels.

A case of poor prognosis metastatic trophoblastic disease resistant to chemotherapy with methotrexate, dactinomycin, and cyclophosphamide is presented. The patient responded promptly to etoposide-based combination chemotherapy and has remained in complete remission since completion of therapy.

The clinical and histologic features of eight cases of carcinoma of the endometrium which developed following radiation therapy for squamous cell carcinoma of the cervix are described. No patient had a well-differentiated tumor and significant myometrial invasion was present in all cases. Three of the eight tumors were papillary serous adenocarcinoma. Five of the eight patients developed recurrent tumor and died of their disease. The risk of endometrial cancer in patients previously radiated for cervical cancer is evaluated.

Vitellogenin production was induced in immature diploid and triploid coho salmon by the weekly injection of 17 beta-estradiol at 1 mg/kg body wt. There was no significant difference between diploids and triploids for any of the results obtained, i.e., change in plasma vitellogenin and gonadotropin levels, hepatosomatic index, or pituitary gonadotropin content. Plasma vitellogenin levels were significantly higher in 17 beta-estradiol-treated fish than in sham-injected fish within a week of the first injection, and continued to rise with each subsequent injection. Plasma gonadotropin levels, on the other hand, were slightly (but significantly) depressed. The 17 beta-estradiol-treated fish had higher hepatosomatic indices and pituitary gonadotropin contents than sham-injected fish by 3 weeks after the first treatment. These data suggest that the occasional postmeiotic oocytes observed in triploids do not grow to full maturity due, in part, to an absent or diminished estrogen stimulus from the ovary on hepatic vitellogenin production.

Patient-controlled analgesia (PCA) is currently being evaluated as an alternative to prn intramuscular injections for the relief of postoperative pain in patients with gynecologic malignancies. From June 1985 to May 1986, twenty patients undergoing abdominal hysterectomy received PCA rather than traditional intramuscular injections for relief of postoperative pain. PCA was administered by a lightweight, wearable, disposable system, the Travenol Infuser with Patient Control Module. The PCA device delivered a 1-mg intermittent intravenous injection of morphine upon patient demand with a delay of 6 min between allowable administrations. The mean amount of morphine utilized postoperatively by the PCA patients (66 mg over 48 hr) was not significantly different from the amount used by a control group receiving prn im morphine injections. No cases of respiratory or cardiac depression were observed, and patients were generally alert throughout the period of analgesia. These data suggest that PCA is a safe and effective mode of analgesia administration. In addition, the unique characteristics of this new compact device were well accepted.

Prognostic parameters were evaluated in 22 patients with small (less than or equal to 2 cm) superficially invasive (less than 5 mm) squamous cell carcinoma of the vulva. Primary surgery included radical vulvectomy with bilateral superficial and deep inguinal lymph node dissection in 11 patients, and wide local excision with ipsilateral superficial inguinal lymph node dissection in 11 patients. Of the 22 patients studied, only 2 (9%) had lymph node metastases. Both patients had a single positive ipsilateral superficial inguinal node. Perineural invasion was strongly associated with lymph node metastases (P less than 0.01). In this group of patients, grade, depth of invasion, lymph-vascular space invasion, and lymphoplasmacytic infiltration were not predictive of lymph node metastases (P greater than 0.05). Two patients initially treated with wide local excision and ipsilateral superficial inguinal lymph node dissection developed recurrent vulvar neoplasia on the contralateral vulva, and both were successfully retreated by wide local excision. All patients are presently alive and well with no evidence of disease. None of the histomorphologic parameters studied were predictive of tumor recurrence. These data suggest that wide local excision with ipsilateral superficial inguinal lymphadenectomy is effective in the treatment of patients with small, superficially invasive carcinomas of the vulva.

Plasma gonadotropin (GtH) levels and state of oocyte development were determined in adult female coho salmon following single intraperitoneal injections of LH-RH or its superactive analog des-Gly10[D-Ala6]LH-RH-ethylamide (LH-RHA DAla6). The peptides injected at dosages of 1.0 or 0.2 mg LH-RH and 0.2 or 0.02 mg LH-RHA DAla6/kg bw elevated plasma GtH by 1.5 hr postinjection. The response to the analog was of longer duration. Plasma GtH levels returned to basal levels 24 hr following LH-RH injection while elevated plasma GtH levels were maintained for at least 96 hr in response to the analog. Only those fish injected with LH-RHA DAla6 showed an accelerated rate of germinal vesicle breakdown (GVBD). Based on their relative effects on GVBD, LH-RHA DAla6 has at least 50 times the biological potency of LH-RH in adult female coho salmon.

17 beta-Estradiol, testosterone, and 17 alpha, 20 beta dihydroxy-4-pregnen-3-one (17 alpha 20 beta P) levels were measured in plasma samples obtained from coho salmon (Oncorhynchus kisutch) during the preovulatory period and following the injection of mammalian gonadotropin releasing hormones. Spontaneous reproductive activity was characterized by a rapid decline in plasma 17 beta-estradiol 10 days prior to ovulation and a large increase in plasma 17 alpha 20 beta P 6 days before ovulation. Testosterone levels remained high (greater than 125 ng/ml) throughout the preovulatory period, with a small peak evident 6 days prior to ovulation. Oocyte development was not accelerated in fish injected with mammalian LH-RH, whereas des-Gly10[D-Ala6]LH-RH-ethylamide (LH-RHA DAla6) promoted germinal vesicle breakdown (GVBD) in 10 out of 14 fish within 96 hr. Only LH-RHA DAla6-injected fish which completed GVBD displayed the characteristic steroid changes observed during spontaneous reproductive activity. In LH-RH-injected fish, there was a transient increase in plasma 17 alpha 20 beta P levels which persisted for less than 24 hr. LH-RHA DAla6-injected fish which failed to complete GVBD maintained high 17 alpha 20 beta P levels, but the peak concentrations were lower than those in fish which completed GVBD. These fish also maintained high plasma 17 beta-estradiol levels when compared to fish which completed GVBD. The appearance of high plasma 17 alpha 20 beta P levels during spontaneous and LH-RHA DAla6-induced reproductive activity was coincident with the time of GVBD. This finding was consistent with the view that 17 alpha 20 beta P functions as the maturation-inducing steroid in salmonids. The induction of GVBD using gonadotropin-releasing hormones was related to the elevation of plasma gonadotropin levels for greater than 24 hr [G. Van Der Kraak, H. R. Lin, E. M. Donaldson, H. M. Dye, and G. A. Hunter (1983) Gen. Comp. Endocrinol. 49, 470-476] and a decrease in 17 beta-estradiol production.

This paper describes an homologous radioimmunoassay for coho salmon vitellogenin that demonstrates parallel cross-reactivity for plasma vitellogenin of all Pacific salmonids tested (chinook, chum, coho, pink, and sockeye salmon, and cutthroat and rainbow trout), but not for Atlantic salmon or two nonsalmonids: common carp and sablefish. Plasma vitellogenin levels were high in ovulatory female Pacific salmonids (micrograms/ml to mg/ml range), but were mostly nondetectable in spermiating males of the same species.

From November 1, 1987, to July 1, 1988, 506 asymptomatic patients 40 years or older underwent screening vaginal sonography at the University of Kentucky Medical Center. Eligibility requirements included no known pelvic symptoms or clinical abnormalities, no history of pelvic radiation, and no history of ovarian cancer. Each ovary was measured in three planes and ovarian volume was calculated using the prolate ellipsoid formula. Ovarian morphology was classified as uniformly hypoechogenic, cystic, solid, or complex. The upper limit of normal for ovarian volume was 18 cm3 in premenopausal women and 8 cm3 in post-menopausal women. With respect to these criteria, 12 patients (2.4%) were noted to have abnormal sonograms, and 10 agreed to surgery. All 10 patients had ovarian tumors with dimensions equal to those predicted by ultrasound. These tumors included four serous cystadenomas, three endometriomas, two cystic teratomas, and one adenocarcinoma. Vaginal sonography is a relatively simple test that can detect subtle changes in ovarian size and morphology. Further evaluation of this test as a screening method for ovarian cancer should be performed in the setting of controlled clinical trials that emphasize cost control and strict patient follow-up.