Melissa A. Melan

Differential Effects of Endoplasmic Reticulum Stress-induced Autophagy on Cell Survival

Autophagy is a cellular response to adverse environment and stress, but its significance in cell survival is not always clear. Here we show that autophagy could be induced in the mammalian cells by chemicals, such as A23187, tunicamycin, thapsigargin, and brefeldin A, that cause endoplasmic reticulum stress. Endoplasmic reticulum stress-induced autophagy is important for clearing polyubiquitinated protein aggregates and for reducing cellular vacuolization in HCT116 colon cancer cells and DU145 prostate cancer cells, thus mitigating endoplasmic reticulum stress and protecting against cell death. In contrast, autophagy induced by the same chemicals does not confer protection in a normal human colon cell line and in the non-transformed murine embryonic fibroblasts but rather contributes to cell death. Thus the impact of autophagy on cell survival during endoplasmic reticulum stress is likely contingent on the status of cells, which could be explored for tumor-specific therapy.

Genital Tract Shedding of Herpes Simplex Virus Type 2 in Women: Effects of Hormonal Contraception, Bacterial Vaginosis, and Vaginal Group B Streptococcus Colonization

BACKGROUND Genital infections due to herpes simplex virus type 2 (HSV-2) are characterized by frequent reactivation and shedding of the virus and by the attendant risk of transmission to sexual partners. We investigated the effects of vaginal coinfections and hormonal contraceptive use on genital tract shedding of HSV-2 in women. METHODS A total of 330 HSV-2-seropositive women were followed every 4 months for a year. At each visit, one vaginal swab specimen was obtained for detection of HSV-2 by polymerase chain reaction, a second vaginal swab specimen was obtained for detection of group B Streptococcus (GBS) organisms and yeast by culture, and a vaginal smear was obtained for the diagnosis of bacterial vaginosis by Gram staining. RESULTS HSV-2 DNA was detected in 88 (9%) of 956 vaginal swab specimens. Independent predictors of genital tract shedding of HSV-2 were HSV-2 seroconversion during the previous 4 months (adjusted odds ratio [aOR], 3.0; 95% confidence interval [CI], 1.3-6.8), bacterial vaginosis (aOR, 2.3; 95% CI, 1.3-4.0), high-density vaginal GBS colonization (aOR, 2.2; 95% CI, 1.3-3.8), and use of hormonal contraceptives (aOR, 1.8; 95% CI, 1.1-2.8). CONCLUSIONS The present study identifies hormonal contraceptive use, bacterial vaginosis, and high-density vaginal GBS colonization as risk factors for genital tract shedding of HSV-2 in women. Because hormonal contraceptives are used by millions of women worldwide and because bacterial vaginosis and vaginal GBS colonization are common vaginal conditions, even modest associations with HSV-2 shedding would result in substantial attributable risks for transmission of the virus.

The Associations Between Pelvic Inflammatory Disease, Trichomonas vaginalis Infection, and Positive Herpes Simplex Virus Type 2 Serology

Objective: Roles for Chlamydia trachomatis and Neisseria gonorrhoeae infections in pelvic inflammatory disease pathogenesis are well delineated; however, the etiologic contributions of herpes simplex virus type 2 (HSV-2) and Trichomonas vaginalis have been underexplored. Goal: The goal of this study was to investigate the association between acute and plasma cell endometritis, fallopian tube obstruction, HSV-2 serology, and T. vaginalis infection. Study Design: The authors conducted a cross-sectional secondary analysis of 736 women at risk for bacterial sexually transmitted diseases that used endometrial biopsy data obtained at enrollment as well as hysterosalpingography results obtained 12 weeks after enrollment. Results: Women diagnosed with T. vaginalis at enrollment were more likely to have histologic evidence of acute endometritis. Both plasma cell and acute endometritis were significantly more common among women with positive serology HSV-2; furthermore, women coinfected with HSV-2 and C. trachomatis, N. gonorrhoeae, T. vaginalis, or bacterial vaginosis were much more likely to be diagnosed with acute endometritis than were women infected with HSV-2 or one of these pathogens alone. Among women with available HSV-2 serology and hysterosalpingogram results, HSV-2 was the only genital tract pathogen infection associated with fallopian tube obstruction. Conclusions: Our analyses demonstrate that T. vaginalis infection and positive HSV-2 serology are associated with endometritis. Further work will be needed to determine the specific roles these pathogens may play in pelvic inflammatory disease pathogenesis.

Melatonin and breast cancer: cellular mechanisms, clinical studies and future perspectives

Recent studies have suggested that the pineal hormone melatonin may protect against breast cancer, and the mechanisms underlying its actions are becoming clearer. Melatonin works through receptors and distinct second messenger pathways to reduce cellular proliferation and to induce cellular differentiation. In addition, independently of receptors melatonin can modulate oestrogen-dependent pathways and reduce free-radical formation, thus preventing mutation and cellular toxicity. The fact that melatonin works through a myriad of signalling cascades that are protective to cells makes this hormone a good candidate for use in the clinic for the prevention and/or treatment of cancer. This review summarises cellular mechanisms governing the action of melatonin and then considers the potential use of melatonin in breast cancer prevention and treatment, with an emphasis on improving clinical outcomes.

MT1 melatonin receptor internalization underlies melatonin-induced morphologic changes in Chinese hamster ovary cells and these processes are dependent on Gi proteins, MEK 1/2 and microtubule modulation.

Abstract:  Melatonin induces cellular differentiation in numerous cell types. Data show that multiple mechanisms are involved in these processes that are cell‐type specific and may be receptor dependent or independent. The focus of this study was to specifically assess the role of human MT1 melatonin receptors in cellular differentiation using an MT1‐Chinese hamster ovary (CHO) model; one that reproducibly produces measurable morphologic changes in response to melatonin. Using multiple approaches, we show that melatonin induces MT1‐CHO cells to hyperelongate through a MEK 1/2, and ERK 1/2‐dependent mechanism that is dependent upon MT1 receptor internalization, Gi protein activation, and clathrin‐mediated endocytosis. Using immunoprecipitation analysis, we show that MT1 receptors form complexes with Giα 2,3, Gqα, β‐arrestin‐2, MEK 1/2, and ERK 1/2 in the presence of melatonin. We also show that MEK and ERK activity that is induced by melatonin is dependent on Gi protein activation, clathrin‐mediated endocytosis and is modulated by microtubules. We conclude from these studies that melatonin‐induced internalization of human MT1 melatonin receptors in CHO cells is responsible for activating both MEK 1/2 and ERK 1/2 to drive these morphologic changes. These events, as mediated by melatonin, require Gi protein activation and endocytosis mediated through clathrin, to form MT1 receptor complexes with β‐arrestin‐2/MEK 1/2 and ERK 1/2. The MT1‐CHO model is invaluable to mapping out signaling cascades as mediated through MT1 receptors especially because it separates out MEK/ERK 1/2 activation by MT1 receptors from that of receptor tyrosine kinases.

Microtubules modulate melatonin receptors involved in phase-shifting circadian activity rhythms: in vitro and in vivo evidence

Abstract:  MT1 melatonin receptors expressed in Chinese hamster ovary (CHO) cells remain sensitive to a melatonin re‐challenge even following chronic melatonin exposure when microtubules are depolymerized in the cell, an exposure that normally results in MT1 receptor desensitization. We extended our findings to MT2 melatonin receptors using both in vitro and in vivo approaches. Using CHO cells expressing human MT2 melatonin receptors, microtubule depolymerization prevents the loss in the number of high potency states of the receptor when compared to melatonin‐treated cells. In addition, microtubule depolymerization increases melatonin‐induced PKC activity but not PI hydrolysis via Gi proteins similar to that shown for MT1Rs. Furthermore, microtubule depolymerization in MT2‐CHO cells enhances the exchange of GTP on Gi‐proteins using a photoaffinity analog of GTP. To test whether microtubules are capable of modulating melatonin‐induced phase‐shifts, microtubules are depolymerized specifically within the suprachiasmatic nucleus of the hypothalamus (SCN) of the Long Evans rat and the efficacy of melatonin to phase shift their circadian activity rhythms was assessed and compared to animals with intact SCN microtubules. We find that microtubule depolymerization in the SCN using either Colcemid or nocodazole enhances the efficacy of 10 pm melatonin to phase‐shift the activity rhythms of the Long Evans rat. No enhancement occurs in the presence of β‐lumicolchicine, the inactive analog of Colcemid. Taken together, these data suggest that microtubule dynamics can modulate melatonin‐induced phase shifts of circadian activity rhythms which may explain, in part, why circadian disturbances occur in individuals afflicted with diseases associated with microtubule disturbances.

Targeted next-generation sequencing panel (GlioSeq) provides comprehensive genetic profiling of central nervous system tumors

BACKGROUND Identification of genetic changes in CNS tumors is important for the appropriate clinical management of patients. Our objective was to develop a next-generation sequencing (NGS) assay for simultaneously detecting the various types of genetic alterations characteristic for adult and pediatric CNS tumors that can be applied to small brain biopsies. METHODS We report an amplification-based targeted NGS assay (GlioSeq) that analyzes 30 genes for single nucleotide variants (SNVs) and indels, 24 genes for copy number variations (CNVs), and 14 types of structural alterations in BRAF, EGFR, and FGFR3 genes in a single workflow. GlioSeq performance was evaluated in 54 adult and pediatric CNS tumors, and the results were compared with fluorescence in-situ hybridization, Sanger sequencing, and reverse transcription PCR. RESULTS GlioSeq correctly identified 71/71 (100%) genetic alterations known to be present by conventional techniques, including 56 SNVs/indels, 9 CNVs, 3 EGFRvIII, and 3 KIAA1549-BRAF fusions. Only 20 ng of DNA and 10 ng of RNA were required for successful sequencing of 100% frozen and 96% formalin-fixed, paraffin-embedded tissue specimens. The assay sensitivity was 3%-5% of mutant alleles for SNVs and 1%-5% for gene fusions. The most commonly detected alterations were IDH1, TP53, TERT, ATRX. CDKN2A, and PTEN in high-grade gliomas, followed by BRAF fusions in low-grade gliomas and H3F3A mutations in pediatric gliomas. CONCLUSIONS GlioSeq NGS assay offers accurate and sensitive detection of a wide range of genetic alterations in a single workflow. It allows rapid and cost-effective profiling of brain tumor specimens and thus provides valuable information for patient management.

Modulation of melatonin receptors and G-protein function by microtubules

Abstract:  Chronic melatonin exposure produces microtubule rearrangements in Chinese hamster ovary (CHO) cells expressing the human MT1 melatonin receptor while at the same time desensitizing MT1 receptors. Because microtubule rearrangements parallel MT1 receptor desensitization, we tested whether microtubules modulate receptor responsiveness. We determined whether depolymerization of microtubules by Colcemid, which prevents melatonin‐induced outgrowths in MT1‐expressing CHO cells, also prevents MT1 receptor desensitization by affecting G‐GTP exchange on G‐proteins. In this study, we found that depolymerization of microtubules in MT1 receptor expressing cells, prevented melatonin‐induced receptor desensitization reflected by an increase in the number of high potency sites when compared with melatonin‐treated cells. Further examination of the mechanism(s) underlying this desensitization suggested that these effects occurred at the level of G‐proteins. Depolymerization of microtubules during melatonin‐induced desensitization, attenuated forskolin‐induced cAMP accumulation, the opposite of which usually occurs following melatonin exposure alone. Concomitant to this attenuation in the forskolin response was a reduction in the amount of Giα protein coupled to MT1 receptors and an increase in [32P] azidoanilido GTP incorporation into Gi proteins. These data are consistent with the findings that microtubule depolymerization did not affect MT1/Gq coupling nor did it affect melatonin‐induced phosphoinositide hydrolysis following melatonin exposure. However, interestingly, microtubule depolymerization enhanced melatonin‐induced protein kinase C activation that was blocked in the presence of pertussis toxin. These data demonstrate that microtubule dynamics can modulate melatonin receptor function through their actions on Gi proteins and impact on downstream signaling cascades.

Dendritic Cell Activation and Memory Cell Development Are Impaired among Mice Administered Medroxyprogesterone Acetate Prior to Mucosal Herpes Simplex Virus Type 1 Infection

Epidemiological studies indicate that the exogenous sex steroid medroxyprogesterone acetate (MPA) can impair cell-mediated immunity, but mechanisms responsible for this observation are not well defined. In this study, MPA administered to mice 1 wk prior to HSV type 1 (HSV-1) infection of their corneal mucosa impaired initial expansion of viral-specific effector and memory precursor T cells and reduced the number of viral-specific memory T cells found in latently infected mice. MPA treatment also dampened expression of the costimulatory molecules CD40, CD70, and CD80 by dendritic cells (DC) in lymph nodes draining acute infection, whereas coculture of such DC with T cells from uninfected mice dramatically impaired ex vivo T cell proliferation compared with the use of DC from mice that did not receive MPA prior to HSV-1 infection. In addition, T cell expansion was comparable to that seen in untreated controls if MPA-treated mice were administered recombinant soluble CD154 (CD40L) concomitant with their mucosal infection. In contrast, the immunomodulatory effects of MPA were infection site dependent, because MPA-treated mice exhibited normal expansion of virus-specific T cells when infection was systemic rather than mucosal. Taken together, our results reveal that the administration of MPA prior to viral infection of mucosal tissue impairs DC activation, virus-specific T cell expansion, and development of virus-specific immunological memory.

Molecular Analysis of the Simultaneous Production of Two SHV-Type Extended-Spectrum Beta-Lactamases in a Clinical Isolate of Enterobacter cloacae by Using Single-Nucleotide Polymorphism Genotyping

ABSTRACT Bacteria that simultaneously produce multiple extended-spectrum beta-lactamases are frequently isolated. We report an Enterobacter cloacae isolate, ES24, producing four different beta-lactamases (AmpC type beta-lactamase, TEM-1, SHV-7, and a novel extended-spectrum beta-lactamase, SHV-30). Direct sequencing of blaSHV gene products gave a “double peak” at position 703, suggesting the presence of more than one allele. Using fluorescence resonance energy transfer real-time PCR to detect single-nucleotide polymorphisms, we were able to distinguish two different blaSHV genes in a single isolate. This may prove to be a useful technique in surveys of beta-lactamase production in contemporary clinical isolates.