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Ovarian cancer is the second most common gynaecologic malignancy and the main cause of death from gynaecologic cancer, due to late diagnosis and chemoresistance. Studies have reported the role of cysteine in cancer, by contributing for hydrogen sulphide (H 2 S) generation and as a precursor of glutathione (GSH). However, the role of cysteine in the adaptation to hypoxia and therapy response remains unclear. We used several ovarian cancer cell lines, ES2, OVCAR3, OVCAR8, A2780 and A2780cisR, to clarify cysteine relevance in ovarian cancer cells survival upon hypoxia and carboplatin. Results show that ES2 and OVCAR8 cells presented a stronger dependence on cysteine availability upon hypoxia and carboplatin exposure than OVCAR3 cells. Interestingly, the A2780 cisR, but not A2780 parental cells, benefits from cysteine upon carboplatin exposure, showing that cysteine is crucial for chemoresistance. Moreover, GSH degradation and subsequent cysteine recycling pathway is associated with ovarian cancer as seen in peripheral blood serum from patients. Higher levels of total free cysteine (Cys) and homocysteine (HCys) were found in ovarian cancer patients in comparison with benign tumours and lower levels of GSH were found in ovarian neoplasms patients in comparison with healthy individuals. Importantly, the total and S-Homocysteinylated levels distinguished blood donors from patients with neoplasms as well as patients with benign from patients with malignant tumours. The levels of S-cysteinylated proteins distinguish blood donors from patients with neoplasms and the free levels of Cys in serum distinguish blood from patients with benign tumours from patients with malignant tumours. Herein we disclosed that cysteine contributes for a worse disease prognosis, allowing faster adaptation to hypoxia and protecting cells from carboplatin. The measurement of serum cysteine levels can be an effective tool for early diagnosis, for outcome prediction and follow up of disease progression.

The cellular components of microenvironment are partners of cancer cells, sharing soluble factors and organic molecules to accomplish tumor energy and biomass demands. We tested the role of fibroblasts in fatty acids metabolism in breast cancer, addressing fatty acid synthase (FASN) expression and activity, the expression of lipids chaperons (FABPs) and transporters (FATPs) and lipids cellular content. We showed that the amount of lipids increased in cancer cells exposed to fibroblasts conditioned media, showing that lipids transfer is crucial in this metabolic cross-talk. Accordingly, it was seen in those cancer cells a concomitant decrease in the expression of FABP2 and FABP3 and an increase in FATP1 expression, whose function is independent of FABPs. The in vivo experiment corroborates the role of CAFs in tumor growth. Our study is one more step toward the understanding of metabolic dynamics between cancer cells and CAFs, disclosing FATP1 as a putative target to disturb the transfer of lipids between CAFs and breast cancer cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We have recently described a new class of dendrimers with tryptophan (Trp) on the surface that show dual antiviral activities against HIV and EV71 enterovirus. The prototype compound of this family is a pentaerythritol derivative with 12 Trps on the periphery. Here we complete the structure-activity relationship studies of this family to identify key features that might be significant for the antiviral activity. With this aim, novel dendrimers containing different amino acids (aromatic and non-aromatic), tryptamine (a "decarboxylated" analogue of Trp) and N-methyl Trp on the periphery have been prepared. Dendrimer with N-Methyl Trp was the most active against HIV-1 and HIV-2 while dendrimer with tyrosine was endowed with the most potent antiviral activity against EV71. This tyrosine dendrimer proved to inhibit a large panel of EV71 clinical isolates (belonging to different clusters) in the low nanomolar/high picomolar range. In addition, a new synthetic procedure (convergent approach) has been developed for the synthesis of the prototype and some other dendrimers. This convergent approach proved more efficient (higher yields, easier purification) than the divergent approach previously reported., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: To determine human papillomavirus (HPV) types in invasive cervical cancer in Portugal., Methods: Cases diagnosed at the Instituto Português de Oncologia de Lisboa de Francisco Gentil from the year 1928 to 2005 were selected for HPV DNA detection and genotyping using SPF10/DEIA/LiPA25 system., Results: Of the 1214 samples that were considered appropriate for HPV detection, 714 (58.8%; 95% CI: 56.0-61.6%) were positive for HPV DNA. This detection rate varied being lower in the first 3 decades (31.3%; 50.1%; 46.5%) and higher in the last decades (77.4-95.1%). This difference was due probably to the fixative used in the first three decades. The five most common types identified among HPV positive cases were HPV16 (58.2%), HPV18 (9.2%), HPV33 (6.2%), HPV45 (4.7%) and HPV31 (4.4%). Multiple infections were detected in 2.8% of the cases. HPV16 and 18 accounted for 67.4% of infections. There were no statistically significant changes of these types over the studied period. An increase at patient׳s age at diagnosis was observed in the last decades (p<0.001)., Conclusion: HPV16 and 18 accounts for almost 70% of cervical cancers in all 9 decades studied and support data that effective vaccination against these 2 types will reduce the cervical burden in Portuguese women., (Copyright © 2016. Published by Elsevier B.V.)

Introduction: Uterine carcinosarcoma is a rare and aggressive biphasic malignancy and is currently included in the high risk endometrial carcinoma group. The aims of this study were to determine the clinicopathological profile, treatment, recurrence/progression patterns, survival and prognostic factors., Material and Methods: Retrospective study of 42 patients, surgically staged and followed-up at a cancer centre, between 2005 and 2013. Clinical data was retrieved from records and pathological characteristics were reviewed for this study., Results: Median age was 72 years (61 - 78) and the majority presented comorbid diseases. Stage distribution as follows: 13 (31.0%) stage I; eight (19.0%) stage II; nine (21.4%) stage III; and 12 (28.6%) stage IV. Chemotherapy was instituted in 12 patients and 21 received radiotherapy. Disease progressed in 16 patients and recurred in nine after a short interval. Median overall survival was 18 months (6.8 - 40) and median disease-free survival was 6 months (0 - 22.8). The only independent prognostic factor related with poor survival was serosal invasion (p = 0.02; HR adjusted 4.22; IC 95% 1.29 - 13.79)., Discussion: In accordance to other studies, diagnosis of uterine carcinosarcoma is frequently done with advanced disease and presents a high rate of progression/recurrence. The variable which has been consistently identified as main prognostic factor is stage, but in this study the only independent factor was serosal invasion., Conclusion: The present study represents the larger series of uterine carcinosarcoma studied in Portugal and reflects the clinical presentation, histopathological characteristics and stage at diagnosis and confirms the aggressiveness of this rare tumor.

Epithelial ovarian cancer is the most lethal gynecologic malignancy, despite advances in treatment. The most common histological type, high-grade ovarian serous carcinoma (OSC) is usually diagnosed at an advanced stage, and although these types of tumors frequently respond to surgery and platinum-based chemotherapy, they usually recur. Ovarian clear cell carcinoma (OCCC) is an unusual histological type, which is known to be intrinsically chemoresistant and is associated with poor prognosis in advanced stages. In recent years, genetic alterations and epigenetic modulation of signaling pathways have been reported in OSC and OCCC, including the overexpression of Notch pathway elements and histone deacetylases. Histone deacetylase inhibitors (HDACis), including vorinostat (suberoylanilide hydroxamic acid), alter the transcription of genes involved in cell growth, survival and apoptosis, and have become an attractive therapeutic approach. However, no previous work has addressed the effect of HDACis, and in particular vorinostat, on Notch signaling in ovarian cancer. Therefore, the present study aimed to investigate the modulation of the Notch pathway by vorinostat in ovarian cancer. Using immunofluorescence and quantitative polymerase chain reaction, the present results revealed that vorinostat activated the Notch pathway in OCCC and OSC cell lines, through different Notch ligands. In OCCC, the activation of the Notch pathway appeared to occur through Delta-like (Dll) ligands 1, 2 and 3, whereas in OSC Dll1 and Jagged 1 and 2 ligands were involved. The activation of the Notch pathway by vorinostat, in OCCC and OSC cell lines, culminated in the increased expression of the same downstream transcription factors, hairy enhancer of split ( Hes ) 1 and 5, and Hes -related proteins 1 and 2. In conclusion, vorinostat modulates the expression of several downstream targets of the Notch pathway and independent Notch receptors and ligands that are expressed in OSC and OCCC. This upregulation of the Notch pathway may explain why vorinostat therapy fails in ovarian carcinoma treatment, as shown in certain clinical trials.

Tryptophan dendrimers that inhibit HIV replication by binding to the HIV envelope glycoproteins gp120 and gp41 have unexpectedly also proven to be potent, specific, and selective inhibitors of the replication of the unrelated enterovirus A71. Dendrimer 12, a consensus compound that was synthesized on the basis of the structure-activity relationship analysis of this series, is 3-fold more potent against the BrCr lab strain and, surprisingly, inhibits a large panel of clinical isolates in the low-nanomolar/high-picomolar range., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Chemoresistance to platinum-based antineoplastic agents is a consistent feature among ovarian carcinomas; however, whereas high-grade serous carcinoma (OSC) acquires resistance during chemotherapy, ovarian clear cell carcinoma (OCCC) is intrinsically resistant. The main objective of this study was to explore, in vitro and in vivo, if hepatocyte nuclear factor 1β (HNF1β) and glutaminolysis contribute for the resistance of OCCC to carboplatin through the intrinsically increased GSH bioavailability. To disclose the role of HNF1β, experiments were also performed in an OSC cell line, which does not express HNF1β. Metabolic profiles, GSH quantification, HNF1β, and γ-glutamylcysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM) expression, cell cycle, and death were assessed in ES2 cell line (OCCC) and OVCAR3 cell line (OSC); HNF1β knockdown was performed in ES2 and murine model of subcutaneous and peritoneal OCCC tumors was established to test buthionine sulphoxamine (BSO), as a sensitizer to carboplatin. Glutaminolysis is activated in ES2 and OVCAR3, though ES2 exclusively synthesizes amino acids and GSH. ES2 cells are more resistant to carboplatin than OVCAR3 and the abrogation of GSH production by BSO sensitizes ES2 to carboplatin. HNF1β regulates the expression of GCLC, but not GCLM, and consequently GSH production in ES2. In vivo, BSO prior to carboplatin reduces dramatically subcutaneous tumor size and GSH levels, as well as peritoneal dissemination. Our study discloses HNF1β as the mediator of intrinsic OCCC chemoresistance and sheds a light to re-explore a cancer adjuvant therapeutic approach using BSO to overcome the lack of efficient therapy in OCCC.

Background: We conducted a large international study to estimate fractions of head and neck cancers (HNCs) attributable to human papillomavirus (HPV-AFs) using six HPV-related biomarkers of viral detection, transcription, and cellular transformation., Methods: Formalin-fixed, paraffin-embedded cancer tissues of the oral cavity (OC), pharynx, and larynx were collected from pathology archives in 29 countries. All samples were subject to histopathological evaluation, DNA quality control, and HPV-DNA detection. Samples containing HPV-DNA were further subject to HPV E6*I mRNA detection and to p16(INK4a), pRb, p53, and Cyclin D1 immunohistochemistry. Final estimates of HPV-AFs were based on HPV-DNA, HPV E6*I mRNA, and/or p16(INK4a) results., Results: A total of 3680 samples yielded valid results: 1374 pharyngeal, 1264 OC, and 1042 laryngeal cancers. HPV-AF estimates based on positivity for HPV-DNA, and for either HPV E6*I mRNA or p16(INK4a), were 22.4%, 4.4%, and 3.5% for cancers of the oropharynx, OC, and larynx, respectively, and 18.5%, 3.0%, and 1.5% when requiring simultaneous positivity for all three markers. HPV16 was largely the most common type. Estimates of HPV-AF in the oropharynx were highest in South America, Central and Eastern Europe, and Northern Europe, and lowest in Southern Europe. Women showed higher HPV-AFs than men for cancers of the oropharynx in Europe and for the larynx in Central-South America., Conclusions: HPV contribution to HNCs is substantial but highly heterogeneous by cancer site, region, and sex. This study, the largest exploring HPV attribution in HNCs, confirms the important role of HPVs in oropharyngeal cancer and drastically downplays the previously reported involvement of HPVs in the other HNCs., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The glycoprotein gp120 of the HIV-1 viral envelope has a high content in mannose residues, particularly α-1,2-mannose oligomers. Compounds that interact with these high-mannose type glycans may disturb the interaction between gp120 and its (co)receptors and are considered potential anti-HIV agents. Previously, we demonstrated that a tripodal receptor (1), with a central scaffold of 1,3,5-triethylbenzene substituted with three 2,3,4-trihydroxybenzoyl groups, selectively recognizes α-1,2-mannose polysaccharides. Here we present additional studies to determine the anti-HIV-1 activity and the mechanism of antiviral activity of this compound. Our studies indicate that 1 shows anti-HIV-1 activity in the low micromolar range and has pronounced gp120 binding and HIV-1 integrase inhibitory capacity. However, gp120 binding rather than integrase inhibition seems to be the primary mechanism of antiviral activity of 1., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Dendrimers containing from 9 to 18 tryptophan residues at the peryphery have been efficiently synthesized and tested against HIV replication. These compounds inhibit an early step of the replicative cycle of HIV, presumably virus entry into its target cell. Our data suggest that HIV inhibition can be achieved by the preferred interaction of the compounds herein described with glycoproteins gp120 and gp41 of the HIV envelope preventing interaction between HIV and the (co)receptors present on the host cells. The results obtained so far indicate that 9 tryptophan residues on the periphery are sufficient for efficient gp120/gp41 binding and anti-HIV activity., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Linear and branched compounds that contain two, three or five units of galloyl (3,4,5-trihydroxybenzoyl) or its isomer 2,3,4-trihydroxybenzoyl, as well as other mono- or dihydroxybenzoyl moieties have been synthesized. These molecules have been evaluated for their in vitro inhibitory effects against a wide panel of viruses showing preferential activity against HIV and HCV. Our structure-activity relationship studies demonstrated that the 2,3,4-trihydroxybenzoyl moiety provides better antiviral activities than the galloyl (3,4,5-trihydroxybenzoyl) moiety that is present in natural green tea catechins. This observation can be of interest for the further rational exploration of compounds with anti-HCV/HIV properties. The most notable finding with respect to HIV is that the tripodal compounds 43 and 45, with three 2,3,4-trihydroxybenzoyl moieties, showed higher activities than linear compounds with only one or two. With respect to HCV, the linear compounds, 52 and 41, containing a 12 polymethylene chain and two 2,3 di- or 2,3,4 tri-hydroxybenzoyl groups respectively at the ends of the molecule showed good antiviral efficiency. Furthermore, the anti-HCV activity of both compounds was observed at concentrations well below the cytotoxicity threshold. A representative member of these compounds, 41, showed that the anti-HCV activity was largely independent of the genetic make-up of the HCV subgenomic replicon and cell lines used., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Background: Both primary and secondary gynaecological neuroendocrine (NE) tumours are uncommon, and the literature is scarce concerning their imaging features., Methods: This article reviews the epidemiological, clinical and imaging features with pathological correlation of gynaecological NE tumours., Results: The clinical features of gynaecological NE tumours are non-specific and depend on the organ of origin and on the extension and aggressiveness of the disease. The imaging approach to these tumours is similar to that for other histological types and the Revised International Federation of Gynecology and Obstetrics (FIGO) Staging System also applies to NE tumours. Neuroendocrine tumours were recently divided into two groups: poorly differentiated neuroendocrine carcinomas (NECs) and well-differentiated neuroendocrine tumours (NETs). NECs include small cell carcinoma and large cell neuroendocrine carcinoma, while NETs account for typical and atypical carcinoids. Cervical small cell carcinoma and ovarian carcinoid are the most common gynaecological NE tumours. The former typically behaves aggressively; the latter usually behaves in a benign fashion and tends to be confined to the organ., Conclusion: While dealing with ovarian carcinoids, extra-ovarian extension, bilaterality and multinodularity raise the suspicion of metastatic disease. NE tumours of the endometrium and other gynaecological locations are very rare., Teaching Points: • Primary or secondary neurondocrine (NE) tumours of the female genital tract are rare. • Cervical small cell carcinoma and ovarian carcinoids are the most common gynaecological NE tumours. • Cervical small cell carcinomas usually behave aggressively. • Ovarian carcinoids tend to behave in a benign fashion. • The imaging approach to gynaecological NE tumours and other histological types is similar.

Here we describe the case of a 19-year-old woman with a poorly differentiated ovarian Sertoli-Leydig cell tumor and an elevated serum alpha-fetoprotein level. The patient presented with diffuse abdominal pain and bloating. Physical examination, ultrasound, and magnetic resonance imaging revealed a right ovarian tumor that was histopathologically diagnosed as a poorly differentiated Sertoli-Leydig cell tumor with heterologous elements. Her alpha-fetoprotein serum level was undetectable after tumor resection. Sertoli-Leydig cell tumors are rare sex cord-stromal tumors that account for 0.5% of all ovarian neoplasms. Sertoli-Leydig cell tumors tend to be unilateral and occur in women under 30 years of age. Although they are the most common virilizing tumor of the ovary, about 60% are endocrine-inactive tumors. Elevated serum levels of alpha-fetoprotein are rarely associated with Sertoli-Leydig cell tumors, with only approximately 30 such cases previously reported in the literature. The differential diagnosis should include common alpha-fetoprotein-producing ovarian entities such as germ cell tumors, as well as other non-germ cell tumors that have been rarely reported to produce this tumor marker.

The synthesis and the assessment of the anti-HIV activity of a set of molecules inspired by the multivalent structures of some naturally-occurring polyphenols (tannins) are reported. Different multibranched scaffolds have been derived from pentaerythritol as the central core which distribute spatially synthetic polyphenolic subunits based on 1-substituted 2,3,4-trihydroxyphenyl moieties. A tetrapodal compound () bearing four N-(2,3,4-trihydroxyphenyl)amide groups, exhibits remarkable selective activity against HIV-1 with EC50 values in the micromolar scale, in the same range as those reported for the most representative anti-HIV tannins. Preliminary SAR studies emphasize the importance of the 1-substituted 2,3,4-trihydroxyphenyl moiety, the presence of an amide as the linker and the multivalent architecture of these molecules, since the anti-HIV activity increases with the number of polyphenolic moieties. The data support the interest in synthetic polyphenols and represent a promising starting point for further design and development of selective HIV-1 inhibitors.

Sclerosing polycystic adenosis is a rare salivary gland lesion. Recently, this entity has been regarded as a neoplastic lesion of low-grade malignant potential but, to date, no invasive carcinoma, metastases, or associated mortality have been reported. We report the first case of an invasive carcinoma component in a recurrent sclerosing polycystic adenosis lesion.

Background: Human papillomavirus (HPV) contribution in vulvar intraepithelial lesions (VIN) and invasive vulvar cancer (IVC) is not clearly established. This study provides novel data on HPV markers in a large series of VIN and IVC lesions., Methods: Histologically confirmed VIN and IVC from 39 countries were assembled at the Catalan Institute of Oncology (ICO). HPV-DNA detection was done by polymerase chain reaction using SPF-10 broad-spectrum primers and genotyping by reverse hybridisation line probe assay (LiPA25) (version 1). IVC cases were tested for p16(INK4a) by immunohistochemistry (CINtec histology kit, ROCHE). An IVC was considered HPV driven if both HPV-DNA and p16(INK4a) overexpression were observed simultaneously. Data analyses included algorithms allocating multiple infections to calculate type-specific contribution and logistic regression models to estimate adjusted prevalence (AP) and its 95% confidence intervals (CI)., Results: Of 2296 cases, 587 were VIN and 1709 IVC. HPV-DNA was detected in 86.7% and 28.6% of the cases respectively. Amongst IVC cases, 25.1% were both HPV-DNA and p16(INK4a) positive. IVC cases were largely keratinising squamous cell carcinoma (KSCC) (N=1234). Overall prevalence of HPV related IVC cases was highest in younger women for any histological subtype. SCC with warty or basaloid features (SCC&#95;WB) (N=326) were more likely to be HPV and p16(INK4a) positive (AP=69.5%, CI=63.6-74.8) versus KSCC (AP=11.5%, CI=9.7-13.5). HPV 16 was the commonest type (72.5%) followed by HPV 33 (6.5%) and HPV 18 (4.6%). Enrichment from VIN to IVC was significantly high for HPV 45 (8.5-fold)., Conclusion: Combined data from HPV-DNA and p16(INK4a) testing are likely to represent a closer estimate of the real fraction of IVC induced by HPV. Our results indicate that HPV contribution in invasive vulvar cancer has probably been overestimated. HPV 16 remains the major player worldwide., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We present a case of a postmenopausal woman diagnosed with an ovarian mass containing thyroid follicles and foci of papillary thyroid carcinoma during pathological examination. This patient referred having had a metachronous thyroid malignancy 10 years before. The differential diagnosis between a thyroid malignancy arising from a struma ovarii and a metastatic ovarian tumor originating from thyroid-cancer is challenging. Struma ovarii should be considered when thyroid components are the predominant element or when thyroid malignant tissue is identified within an ovarian lesion. Thyroid carcinoma arising from a struma ovarii is reported to occur in a minority of cases. Of these, papillary carcinoma is the most frequent subtype encountered. Regarding primary thyroid carcinomas, papillary carcinomas have a lower metastatic potential when compared to follicular carcinomas, and most of the metastases occur in the cervical lymph nodes. Ovarian metastases are exceedingly rare and generally associated with widespread disease. However, they must be considered in the presence of previous history of malignant thyroid carcinoma. The authors review the main clinical, imaging and therapeutic aspects of both these entities and present the most likely diagnosis.

Zebrafish (Danio rerio) has recently emerged as a new animal model in neuroendocrinology and behavior (e.g., stress physiology and ecotoxicology studies). In these areas, the concentrations of steroid hormones in the blood are often used to study the endocrinological status of individuals. However, due to the small body size of zebrafish, blood sampling is difficult to perform and the amount of plasma obtained per sample for assaying hormones is very small (ca. 1-5 μL), and therefore most studies have been using whole-body hormone concentrations, which implies sacrificing the individuals and hampers sequential sampling of the same individual. Here a noninvasive method to assay steroid hormones from zebrafish holding-water, based on the fact that steroids are released into the fish holding-water through the gills by passive diffusion, is validated. Cortisol and the androgen 11-ketotestosterone (KT) were measured in water samples and compared to plasma levels in the same individuals. Cortisol released to holding-water correlates positively with plasma concentrations, but there was a lack of correlation between KT water and circulating levels. However, KT levels showed a highly significant sex difference that can be used to noninvasively sex individuals. An ACTH challenge test demonstrated that an induced increase in circulating cortisol concentration can be reliably detected in holding-water levels, hence attesting the responsiveness of holding-water levels to fluctuations in circulating levels.

Nucleoside 1, with an unusual tricyclic carbohydrate moiety, specifically inhibits HIV-1 replication while being inactive against HIV-2 or other (retro) viruses. In an attempt to increase the inhibitory efficacy against HIV-1, and to further explore the structural features required for anti-HIV-1 activity, different types of modifications have been carried out on this prototype compound. These include substitution of the ethoxy group at the C-4″ position by alkoxy groups of different length, branching, conformational freedom or functionalization. In addition, the 4″-ethoxy group has been removed or substituted by other functional groups. The role of the tert-butyldimethylsilyl (TBDMS) group at the 2' position has also been studied by preparing the corresponding 2'-deprotected derivative or by replacing it by other silyl (tert-hexyldimethylsilyl) or acyl (acetyl) moieties. Finally, the thymine of the prototype compound has been replaced by N-3-methylthymine, uracil or thiophenyl. Some of these compounds were endowed with a 6- to 7-fold higher selectivity than the prototype 1. The tricyclic nucleosides here described represent a novel type of selective anti HIV-1 inhibitors, targeted at the HIV-1-encoded reverse transcriptase., (Copyright © 2011 Elsevier B.V. All rights reserved.)

On the basis of the interesting inhibitory properties that lectins show against HIV-replication through their interaction with glycoprotein 120 (gp120), we here describe the design, synthesis, and anti-HIV evaluation of three series of 1,3,5-triazine derivatives (monomers, dimers, and trimers) functionalized with aromatic amino acids meant to mimic interactions that lectins establish with gp120. While monomers were inactive against HIV replication, dimers showed limited anti-HIV activity that is, however, considerably more significant in the trimers series, with EC(50) values in the lower μM range. These findings most likely reflect the requirement of multivalency of the 1,3,5-triazine derivatives to display anti-HIV activity, as lectins do. The pronounced anti-HIV activity (EC(50) ∼ 20 μM) is accompanied by the absence of toxicity in CEM T-cell line (CC(50) > 250 μM). Moreover, SPR experiments revealed that the prototype trimers with a central core of 2,4,6-triethylbenzene and six l-Trp or six l-Tyr residues at the periphery were efficient binders of CXCR4- and CCR5-tropic HIV-1 gp120 (estimated K(D): lower micromolar range). The collected data support the interest of this novel family of anti-HIV agents and qualify them as potential novel microbicide lead compounds.

The diagnosis of parathyroid carcinomas is often difficult. HRPT2 mutations have been identified in familial [hyperparathyroidism-jaw tumor (HPT-JT) syndrome] and sporadic parathyroid carcinomas, supporting that HRPT2 mutations may confer a malignant potential to parathyroid tumors. In this study, we report the clinical, histopathological, and genetic investigation of two unrelated cases, whom had apparently sporadic malignant parathyroid tumors, initially diagnosed as adenomas. In one case, the differential diagnosis was complicated by cervical seeding of parathyroid tumor cells. Genetic studies identified de novo HRPT2 germline mutations in cases 1 (c.518&#95;521delTGTC [p.Ser174LysfsX27]) and 2 (c.226 C > T [p.Arg76X]), unveiling the hereditary HPT-JT syndrome in both patients. Furthermore, the identification of somatic mutations in the patients‟ parathyroid tumors provided evidence for complete inactivation of the HRPT2 gene, which was consistent with the tumor malignant features. The sensitivity of parafibromin immunostaining to detect HRPT2 mutations was limited. The present data suggests that patients with apparently sporadic parathyroid carcinomas, or parathyroid tumors with atypical histological features, should undergo molecular genetic testing, as it may detect germline HRPT2 mutations. Establishing the diagnosis of hereditary HPT-JT syndrome is relevant for clinical counseling and management of the carriers and their relatives.

The development of the Drosophila melanogaster wing depends on its subdivision into anterior and posterior compartments, which constitute two independent cell lineages since their origin in the embryonic ectoderm. The anterior-posterior compartment boundary is the place where signaling by the Hedgehog pathway takes place, and this requires pathway activation in anterior cells by ligand expressed exclusively in posterior cells. Several mechanisms ensure the confinement of hedgehog expression to posterior cells, including repression by Cubitus interruptus, the co-repressor Groucho and Master of thick veins. In this work we identified Kismet, a chromodomain-containing protein of the SNF2-like family of ATPases, as a novel component of the hedgehog transcriptional repression mechanism in anterior compartment cells. In kismet mutants, hedgehog is ectopically expressed in a domain of anterior cells close to the anterior-posterior compartment boundary, causing inappropriate activation of the pathway and changes in the development of the central region of the wing. The contribution of Kismet to the silencing of hedgehog expression is limited to anterior cells with low levels of the repressor form of Cubitus interruptus. We also show that knockdown of CHD8, the kismet homolog in Xenopus tropicalis, is also associated with ectopic sonic hedgehog expression and up-regulation of one of its target genes in the eye, Pax2, indicating the evolutionary conservation of Kismet/CHD8 function in negatively controlling hedgehog expression., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The development of the Drosophila melanogaster wing depends on the correct regulation of cell survival, growth, proliferation, differentiation, and pattern formation. These processes, and the genes controlling then, are common to the development of epithelia in many different organisms. To identify additional genes contributing to wing development we have carried out a genetic screen in mosaic wings carrying clones of homozygous mutant cells. We obtained 12 complementation groups corresponding to genes with a proven role in wing formation such as smoothened, thick veins, mothers against dpp, expanded, and fat and 71 new complementation groups affecting the pattern of veins and the size of wing. We mapped one of these groups to the mediator15 gene (med15), a component of the Mediator complex. We show that Med15 and other members of the Mediator complex are required, among other processes, for the transcription of decapentaplegic target genes.

We report for the first time a case of 2.5 cm polypoid cervical endometriosis with a superficial growth pattern in a 48-year-old patient with past tubal ligation. The lesion showed metaplastic changes (clear cell, eosinophilic, micropapillary) and a prominent Arias Stella reaction in the absence of concomitant pregnancy but presumably related to phytoestrogenic treatment. The eutopic endometrium, however, had a usual proliferative appearance, implying that it showed a different response from the endometriotic tissues, suggesting the possibility of a metaplastic origin for the endocervical polypoid endometriosis. The unusual histology of the lesion led to an erroneous diagnosis of papillary serous carcinoma in the biopsy. This was subsequently excluded on finding endometrial-type stroma surrounding glands, and was confirmed immunohistochemically by a low Ki-67 index and negativity for p53.

An alpha hydroxy pyrrolidine tricyclic nucleoside 3 and its spontaneous reaction with acetone is described. In this transformation highly functionalized polycyclic nucleosides with rather unusual molecular skeletons are formed in a complete regio- and stereoselective way. The reaction involves the formation of three new bonds, two of them novel carbon-carbon bonds, in a one-pot way. An enamine-iminium mechanism with participation of carbinolamine, iminium ion, and enamine intermediates is proposed as a plausible explanation for this transformation. The scope of the reaction is briefly studied concluding that the nature of the ketone (R(1)COR(2)) is critical for the initial attack of the NH to the carbonyl group.

Gene expression is regulated in part by protein complexes containing ATP-dependent chromatin-remodelling factors of the SWI/SNF family. In Drosophila there is only one SWI/SNF protein, named Brahma, which forms the catalytic subunit of two complexes composed of different proteins. The protein Osa defines the BAP complex, and the proteins Polybromo and Bap170 are only present in the complex named PBAP. In this work we have analysed the functional requirements of Osa during Drosophila wing development, and found that osa is needed for cell growth and survival in the wing imaginal disc, and for the correct patterning of sensory organs, veins and the wing margin. Other members of the BAP complex, such as Snr1, Bap55, Mor and Brm, also share these functions of Osa. We focused on the requirement of Osa during the formation of the wing veins. Genetic interactions between osa alleles and mutations affecting the activity of the EGFR pathway suggest that one aspect of Osa is intimately related to the response to EGFR activity. Thus, loss of osa and EGFR signalling results in similar wing vein phenotypes, and osa alleles enhance the loss of veins caused by reduced EGFR activity. In addition, Osa is required for the expression of several targets of EGFR signalling, such as Delta, rhomboid and argos. We suggest that one role of Osa and Brm in the wing is to establish a chromatin environment in the regulatory regions of EGFR target genes, making them available for both activators and repressors and facilitating transcription in response to EGFR signalling.

Primary invasive squamous cell carcinoma of the vagina is rare, and the role of human papilloma virus in its pathogenesis remains unclear. The aims of our study were to determine the distribution of human papilloma virus genotypes in 21 cases of primary invasive squamous cell carcinoma of the vagina and to correlate human papilloma virus genotype with histological subtypes. Patients' clinical records were reviewed for demographic data and the stage of the disease. Tumors (n=21) were classified according to the World Health Organization criteria. Human papilloma virus genotyping (INNO-LiPA HPV Genotyping) was performed in the whole series, and statistical analysis was performed with Fisher's Exact Test and with Student's t-test. The patients' age ranged from 36 to 88 (mean 65) years. Six cases were keratinizing squamous cell carcinoma, and 15 cases were non-keratinizing squamous cell carcinoma (seven non-keratinizing not otherwise specified, three basaloid, and five warty types). The median age of patients with keratinizing squamous cell carcinoma was 73.8 years and that of non-keratinizing squamous cell carcinoma patients was 61.5 years (P=0.08). Human papilloma virus DNA was detected in 17 cases (81%): 13 non-keratinizing squamous cell carcinoma (87%) and four keratinizing squamous cell carcinoma (67%) (P=0.31). The human papilloma virus genotypes identified were: 6, 11, 16, 18, 31, 33, 35, 40, and 58, with human papilloma virus 16 DNA the most prevalent (33%). Invasive squamous cell carcinoma of the vagina is frequently associated with human papilloma virus infection, and human papilloma virus 16 is the most common genotype. Although without statistical significance, keratinizing squamous cell carcinoma is more frequent in older patients, whereas non-keratinizing squamous cell carcinoma more frequently affects younger women. All studied histological subtypes are strongly associated with human papilloma virus infection.

Novel derivatives of the anti-HIV-1 agent, TSAO-T, bearing at the N-3 position alkylating groups or photoaffinity labels were prepared and evaluated for their anti-HIV activity. All of these compounds demonstrated pronounced anti-HIV-1 activity and inhibited HIV-1 RT; however, we were unable to detect stable covalent linkages between inhibitor and enzyme. In addition, compounds with an alcohol functional group connected to the N-3 position through a cis or trans double bond have been prepared. These compounds have been useful to study how the conformational restriction of the linker affects in the interaction between the N-3 substituent and the HIV-1 RT enzyme.

The appropriately substituted 5-O-tosyl derivative (1), easily prepared from 1,2-O-isopropylidene-alpha-d-xylofuranose, serves as a useful precursor for the preparation of highly condensed cyclic carbohydrates. The synthesis involves a first cyclization of the 5-O-tosyl sugar derivative 1 to a highly reactive cyclic enamine, which subsequently undergoes the nucleophilic attack of a bifunctional reagent X(CH2)nZ in a regio- and stereospecific way. Finally, a spontaneous cyclization step allows the formation of a stereochemically defined extra ring, fused to the sugar backbone. The functionalization and size of this ring can be varied by the proper choice of the bifunctional reagent. X-ray diffraction analysis and intensive NMR studies with one of these carbohydrates were performed to highlight the strained nature of these compounds.

The commercially available carbohydrate 1,2-O-isopropylidene-alpha-D-xylofuranose was efficiently transformed into the high-added-value synthetic scaffold 8. The transformation requires the synthesis of the 5-O-tosyl derivative 7 and its subsequent intramolecular cyclization under basic conditions to give the cyclic enamine 8. Reaction of 8 with O-, N-, S-, and C-nucleophiles and amino acids allowed its efficient transformation (one-step, high yields, and easy purifications) into fused cyclic sugar derivatives with rather unusual molecular skeletons in a completely regio- and stereoselective manner. The characteristics of the sugar derivative 8 established here, high reactivity, synthetic accessibility, and the potential for conversion into a vast collection of products by the action of different nucleophiles, indicate that it will prove to be a useful chiral intermediate for achieving skeletal diversity. The constrained structures and dense functionalization of the polycyclic sugar derivatives generated from 8 make these compounds promising candidates for use as starting agents for the production of new analogues and as drugs.

The genome of human immunodeficiency virus type 1 (HIV-1) encodes 15 distinct proteins, three of which provide essential enzymatic functions: a reverse transcriptase (RT), an integrase (IN), and a protease (PR). Since these enzymes are all homodimers, pseudohomodimers or multimers, disruption of protein-protein interactions in these retroviral enzymes may constitute an alternative way to achieve HIV-1 inhibition. A growing number of dimerization inhibitors for these enzymes is being reported. This mini review summarizes some approaches that have been followed for the development of compounds that inhibit those three enzymes by interfering with the dimerization interfaces between the enzyme subunits.

Here we describe a novel type of enzyme-based prodrug approach in which a dipeptide moiety is linked to a nonpeptidic therapeutic drug through an amide bond which is specifically cleaved by the dipeptidyl-peptidase IV (DPP IV/CD26) enzyme activity present in plasma and on the surface of certain cells. DPP IV has high substrate selectivity for peptides with a proline (or an alanine) at the penultimate amino acid position at the N-terminus but tolerates a wide range of natural amino acids at the amino terminal end. A variety of dipeptidyl amide prodrugs of anti-HIV TSAO molecules were synthesized and evaluated for their ability to act as substrates for the enzyme. Our data revealed that DPP IV/CD26 can efficiently recognize such prodrugs as substrates, releasing the parent compound. Moreover, it is possible to modify the half-life and the lipophilicity of the prodrugs by changing the nature of the dipeptide. All conjugates have shown marked in vitro antiviral activities irrespective the the nature of the terminal and/or the penultimate amino acid moiety.

The polymerase activity of HIV-1 reverse transcriptase (RT) is entirely dependent on the heterodimeric structure of the enzyme. Accordingly, RT dimerization represents a target for the development of a new therapeutic class of HIV inhibitors. We previously demonstrated that the N-3-ethyl derivative of 2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5' '-(4' '-amino-1' ',2' '-oxathiole-2' ',2' '-dioxide)thymine (TSAO-T) destabilizes the inter-subunit interactions of HIV-1 RT [Sluis-Cremer, N.; Dmietrinko, G. I.; Balzarini, J.; Camarasa, M.-J.; Parniak, M. A. Biochemistry 2000, 39, 1427-1433]. In the current study, we evaluated the ability of 64 TSAO-T derivatives to inhibit RT dimerization using a novel screening assay. Five derivatives were identified with improved activity compared to TSAO-T. Four of these harbored hydrophilic or aromatic substituents at the N3 position. Furthermore, a good correlation between the ability of the TSAO-T derivatives to inhibit RT dimerization and the enzyme's polymerase activity was also observed. This study provides an important framework for the rational design of more potent inhibitors of RT dimerization.

Scarce examples of exceptionally mild desilylation of tert-butyldimethylsilyl (TBDMS) ether groups by neighboring group participation have been previously described. Here, we investigate, in detail, the discovery of the unusual lability of the 5'-TBDMS group on 4' '-acylamino TSAO derivatives in DMSO solution. The synthesis and comparative chemical stability studies in different solvents of a variety of 4' '-substituted TSAO derivatives bearing different carbonyl functionalities are reported. Modifications have also been performed at the 5'-position of the TSAO molecule to gain insight into the structural requirements for the desilylation to occur. The role of the solvent has also been studied. Additionally, NMR and theoretical investigations have been carried out to get further insight into the conformational, geometric, and/or electronic parameters that may play a role in the "spontaneous" release of the 5'-TBDMS group. A silyl hydrolysis mechanism involving neighboring group participation of the 4' '-acylamino group is proposed.

Novel derivatives of the anti-HIV-1 agent, TSAO-T, bearing at the N-3 position a variety of polar, lipophilic, or aromatic groups linked to that position through flexible polymethylene linkers of different length, were prepared and evaluated for their anti-HIV activity. Several compounds (within the series of polar bearing groups) exhibited a 2-6-fold improved antiviral potency with regard to the lead compound, TSAO-T. Moreover, some of the most active N-3 TSAO derivatives retain antiviral activity against the TSAO-T-resistant HIV-1 strain (Glu138 --> Lys). Interestingly, the N-methylcarboxamide derivative 17 was 5- to 6-fold more active (IC50: 0.56 microM) against recombinant HIV-1 reverse transcriptase than the lead compound, thus becoming the most active TSAO derivative synthesized so far. On the other hand, the N-3 methylcarboxamide TSAO derivative 12 turned out to have the highest selectivity index yet reported for this class of compounds (around > or =12 000).

The coumarins represent a unique class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that were isolated from tropical plants. (+)-Calanolide A, the most potent compound of this class, selects for the T139I resistance mutation in HIV-1 reverse transcriptase (RT). Seven RTs mutated at amino acid position 139 (Ala, Lys, Tyr, Asp, Ile, Ser, and Gln) were constructed by site-directed mutagenesis. The mutant T139Q enzyme retained full catalytic activity compared with wild-type RT, whereas the mutant T139I, T139S, and T139A RTs retained only 85 to 50% of the activity. Mutant T139K, T139D, and T139Y RTs had seriously impaired catalytic activities. The mutations in the T139I and T139D RTs were shown to destabilize the RT heterodimer. (+)-Calanolide A lost inhibitory activity (up to 20-fold) against the mutant T139Y, T139Q, T139K, and T139I enzymes. All of the mutant enzymes retained marked susceptibility toward the other NNRTIs, including nevirapine, delavirdine, efavirenz, thiocarboxanilide UC-781, quinoxaline GW867420X, TSAO [[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)] derivatives, and the nucleoside inhibitor, ddGTP. The fact that the T139I RT 1) proved to be resistant to (+)-calanolide A, 2) represents a catalytically efficient enzyme, and 3) requires only a single transition point mutation (ACA-->ATA) in codon 139 seems to explain why mutant T139I RT virus strains, but not virus strains containing other amino acid changes at this position, predominantly emerge in cell cultures under (+)-calanolide A pressure.

New [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-d-ribofuranosyl]-3'-spiro-5' '-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide) (TSAO) derivatives substituted at the 4' '-amino group of the spiro moiety with different carbonyl functionalities have been designed and synthesized. Various synthetic procedures, on the scarcely studied reactivity of the 3'-spiroaminooxathioledioxide moiety, have been explored. The compounds were evaluated for their inhibitory effect on both wild-type and TSAO-resistant HIV-1 strains, in cell culture. The presence of a methyl ester (10) or amide groups (12) at the 4''-position conferred the highest anti-HIV-1 activity, while the free oxalyl acid derivative (11) was 10- to 20-fold less active against the virus. In contrast, the presence at this position of (un)substituted ureido or acyl groups markedly diminished or annihilated the anti-HIV-1 activity. Surprisingly, some of the target compounds also showed inhibition of human cytomegalovirus (HCMV) replication at subtoxic concentrations. This has never been observed previously for TSAO derivatives. In particular, compound 26 represents the first TSAO derivative with dual anti-HIV-1 and -HCMV activity.

The combination of different anti-HIV agents has become the standard of care for AIDS or HIV-infected individuals. Important progress has been made in the development of drugs for the clinical treatment of HIV infection. To date, 20 drugs have been approved for the treatment of AIDS. However, viral rebound during therapy, the emergence of HIV drug resistance and the need for long-term treatment modalities are the main causes for the failure of current antiretroviral therapy. There is still a need for the development of new drugs that are either less toxic, active against the growing number of drug-resistant HIV strains or directed to novel targets in the viral life cycle. Eleven of the approved anti-HIV drugs target the reverse transcriptase (RT). Among the so-called non-nucleoside RT inhibitors (NNRTIs) TSAO derivatives are an unusual class of compounds that exert their unique selectivity for HIV-1 through a specific interaction with the p51 subunit of HIV-1 RT. They are the only NNRTIs for which amino acids at both subunits (p66 and p51) of HIV-1 RT are needed for optimal interaction with the enzyme. Moreover, the TSAO compounds are the first non-peptide molecules that interfere with the dimerization of the enzyme.

We report here the efficient regio- and stereoselective synthesis of new polycyclic nucleosides using a common cyclic enamine (7) as the starting material. In fact, the reaction of 7, easily prepared by reaction of 5'-O-Tosyl TSAO-T under basic nonnucleophilic conditions (potassium carbonate), with different classes of nucleophiles, for example, nitrogen-, oxygen-, sulfur-, and carbon-based nucleophiles, or with amino acids afforded, with total regio- and stereoselectivity, new bi-, tri-, and tetracyclic nucleosides. This straighforward route represents an original and unambiguously regio- and stereoselective pathway to these compounds. Some of these polycyclic nucleosides may be useful intermediates for a second series of reactions that may lead to the generation of structurally new nucleosides.

Emergence of drug-resistant viral strains is one of the major milestones and the main cause for the failure of antiretroviral therapy. Combination of different anti-HIV agents has become the standard clinical practice to keep the viral load at low or even undetectable levels and to prevent emergence of virus-drug resistance. Among the human immunodeficiency virus (HIV) reverse transcriptase (RT) inhibitors, the so called nonnucleoside RT inhibitors (NNRTIs) have gained a definitive place in the treatment of HIV infections in combination with nucleoside analogue RT inhibitors (NRTIs) and HIV protease inhibitors (PIs). The virus can be markedly suppressed for a relatively long period of time when exposed to multiple drug combination therapy (highly active antiretroviral therapy, HAART). TSAO derivatives are a peculiar group of highly functionalized nucleosides that belong to the so-called nonnucleoside RT inhibitors (NNRTIs). They exert their unique selectivity for HIV-1 through a specific interaction with the p51 subunit of HIV-1 RT. They are the first small molecules that seem to interfere with the dimerization process of the enzyme. This review covers the work carried out with this unique class of specific inhibitors of HIV-1 reverse transcriptase, including structure activity relationship studies (SAR), its mechanism of action, resistance studies, model of interaction with the enzyme, etc.

Two series of analogues of the novel human mitochondrial thymidine kinase inhibitor 1-[(Z)-4-(triphenylmethoxy)-2-butenyl]thymine were synthesized by replacing the triphenylmethoxy moiety by a variety of substituted amines and carboxamides. In all the cases, the selectivity against the mitochondrial enzyme was either maintained or improved, and several derivatives were almost as potent as the parent compound. A molecular model was built that can account for the observed selectivities.

We have previously reported the discovery and preliminary structure-activity relationships of a new class of specific HIV-1 reverse transcriptase (RT) inhibitors whose prototype compound is the 1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3-N-[(carboxy) methyl]-thymine. In an attempt to increase the inhibitory efficacy against HIV-1 RT of this new class of nucleosides, and to further explore the structural features required for anti-HIV-1 activity, different types of modifications have been carried out on the prototype compound. These include substitution of the tert-butyldimethylsilyl groups by other liphophilic groups, replacement of the carboxy group at the N-3 position of the nucleobase by other functional groups, change in the length of the spacer between the thymine and the carboxylic acid residue and substitution of the thymine moiety by other pyrimidine (uracil, 5-ethyluracil) or purine (hypoxanthine) nucleobases. In addition, the most salient structural features of this new class of HIV-1-specific nucleosides have been incorporated into classical HIV RT nucleoside inhibitors such as ddl, AZT, d4T. Our studies demonstrate that both the carboxymethyl moiety at the nucleobase and tert-butyldimethylsilyl groups at the sugar are important structural components since deletion of either of them is detrimental to the antiviral activity.

Reaction of 5'-O-tosyl TSAO-m(3)T (1) with amines has led to the synthesis of new classes of bi- and tricyclic nucleosides. Full details about the synthesis of these compounds and a plausible mechanism to explain their obtention are reported. In addition, we describe the development of a second, more efficient, and higher yielding synthetic route as a general approach for the synthesis of some of these bicyclic nucleosides. To study the conformational behavior of the bi- and tricyclic nucleosides described in this paper, intensive NMR investigations and molecular modeling studies were performed. Conformational analysis indicates that the furanose ring of the compounds described here prefers the eastern side of the pseudorotation cycle with the base substituents preferentially in the anti range. The torsion angle gamma describing the C-4'[bond]C-5' is found to prefer the +sc range. These compounds represent a novel class of E-type conformationally restricted bicyclic ribo-nucleosides containing a [3.3.0]-fused carbohydrate moiety. The bicyclic nucleosides described herein present an interesting potential for diverse and selective chemical treatments on the 2'-hydroxyl and/or the functionalities incorporated at the bridge between C-3' and C-5'.

Hyalinizing clear cell carcinoma (HCCC) is a recently described low-grade carcinoma of the salivary glands presenting two main histological features: clear neoplastic cells and prominent hyalinized stroma. Our aim was to characterize the composition of the hyalinized stroma in three cases of HCCC. An immuno-histochemical study using laminin and type I, III and IV collagens, fibronectin and tenascin antibodies was performed. In one case, electron microscopy study was also done. Collagen I and fibronectin were always present in tumoral stroma. Collagen III was documented in two cases and tenascin at the invasive front of the neoplasm. Collagen IV and laminin were found around neoplastic cells but not at the interstitial stroma. Foci of basal lamina reduplication and large deposits of long space collagen were observed in EM. This study demonstrates that the hyalinized stroma of HCCC is not formed by the accumulation of basement membrane-type material since it is mostly composed by collagen I and fibronectin.

Salivary gland-type carcinomas arising in Bartholin's gland are rare neoplasms. We report the case of a 75-year-old female who presented with a vulvar tumor with morphological and immunophenotypical features identical to those of salivary gland basal cell adenocarcinomas. We believe that the tumor was most likely of Bartholin's gland origin, although no Bartholin's gland tissue was found adjacent to the neoplasm. This type of malignancy with myoepithelial differentiation has not been previously reported to arise in the Bartholin's gland.