Fetal hemoglobin induction in azacytidine responders enlightens methylation patterns related to blast clearance in higher-risk MDS and CMML

Background: As new treatment options for patients with higher-risk myelodysplastic syndromes are emerging, identification of prognostic markers for hypomethylating agent (HMA) treatment and understanding mechanisms of their delayed and short-term responses are essential. Early fetal hemoglobin (HbF) induction has been suggested as a prognostic indicator for decitabine-treated patients. Although epigenetic mechanisms are assumed, responding patients’ epigenomes have not been thoroughly examined. We aimed to clarify HbF kinetics and prognostic value for azacytidine treated patients, as well as the epigenetic landscape that might influence HbF re-expression and its clinical relevance. Results: Serial HbF measurements by high-performance liquid chromatography (n = 20) showed induction of HbF only among responders (p = 0.030). Moreover, HbF increase immediately after the first azacytidine cycle demonstrated prognostic value for progression-free survival (PFS) (p = 0.032, HR = 0.19, CI 0.24–1.63). Changes in methylation patterns were revealed with methylated DNA genome-wide sequencing analysis (n = 7) for FOG-1, RCOR-1, ZBTB7A and genes of the NuRD-complex components. Targeted pyrosequencing methodology (n = 28) revealed a strong inverse correlation between the degree of γ-globin gene (HBG2) promoter methylation and baseline HbF levels (p = 0.003, rs = − 0.663). A potential epigenetic mechanism of HbF re-expression in azacytidine responders was enlightened by targeted methylation analysis, through hypomethylation of site -53 of HBG2 promoter (p = 0.039, rs = − 0.504), which corresponds to MBD2-NuRD binding site, and to hypermethylation of the CpG326 island of ZBTB7A (p = 0.05, rs = 0.482), a known HbF repressor. These changes were associated to blast cell clearance (pHBG2 = 0.011, rs = 0.480/pZBTB7A = 0.026, rs = 0.427) and showed prognostic value for PFS (pZBTB7A = 0.037, HR = 1.14, CI 0.34–3.8). Conclusions: Early HbF induction is featured as an accessible prognostic indicator for HMA treatment and the proposed potential epigenetic mechanism of HbF re-expression in azacytidine responders includes hypomethylation of the γ-globin gene promoter region and hypermethylation of the CpG326 island of ZBTB7A. The association of these methylation patterns with blast clearance and their prognostic value for PFS paves the way to discuss in-depth azacytidine epigenetic mechanism of action. Graphical abstract: (Figure presented.)</p

Construction, Complete Sequence, and Annotation of a BAC Contig Covering the Silkworm Chorion Locus

The silkmoth chorion was studied extensively by F.C. Kafatos’ group for almost 40 years. However, the complete structure of the chorion locus was not obtained in the genome sequence of Bombyx mori published in 2008 due to repetitive sequences, resulting in gaps and an incomplete view of the locus. To obtain the complete sequence of the chorion locus, expressed sequence tags (ESTs) derived from follicular epithelium cells were used as probes to screen a bacterial artificial chromosome (BAC) library. Seven BACs were selected to construct a contig which covered the whole chorion locus. By Sanger sequencing, we successfully obtained complete sequences of the chorion locus spanning 871,711 base pairs on chromosome 2, where we annotated 127 chorion genes. The dataset reported here will recruit more researchers to revisit one of the oldest model systems which has been used to study developmentally regulated gene expression. It also provides insights into egg development and fertilization mechanisms and is relevant to applications related to improvements in breeding procedures and transgenesis

A Comprehensive Analysis of the Chorion Locus in Silkmoth

Despite more than 40 years of intense study, essential features of the silkmoth chorion (eggshell) are still not fully understood. To determine the precise structure of the chorion locus, we performed extensive EST analysis, constructed a bacterial artificial chromosome (BAC) contig, and obtained a continuous genomic sequence of 871,711 base pairs. We annotated 127 chorion genes in two segments interrupted by a 164 kb region with 5 non-chorion genes, orthologs of which were on chorion bearing scaffolds in 4 ditrysian families. Detailed transcriptome analysis revealed expression throughout choriogenesis of most chorion genes originally categorized as “middle”, and evidence for diverse regulatory mechanisms including cis-elements, alternative splicing and promoter utilization, and antisense RNA. Phylogenetic analysis revealed multigene family associations and faster evolution of early chorion genes and transcriptionally active pseudogenes. Proteomics analysis identified 99 chorion proteins in the eggshell and micropyle localization of 1 early and 6 Hc chorion proteins

Versican but not decorin accumulation is related to malignancy in mammographically detected high density and malignant-appearing microcalcifications in non-palpable breast carcinomas

<p>Abstract</p> <p>Background</p> <p>Mammographic density (MD) and malignant-appearing microcalcifications (MAMCs) represent the earliest mammographic findings of non-palpable breast carcinomas. Matrix proteoglycans versican and decorin are frequently over-expressed in various malignancies and are differently involved in the progression of cancer. In the present study, we have evaluated the expression of versican and decorin in non-palpable breast carcinomas and their association with high risk mammographic findings and tumor characteristics.</p> <p>Methods</p> <p>Three hundred and ten patients with non-palpable suspicious breast lesions, detected during screening mammography, were studied. Histological examination was carried out and the expression of decorin, versican, estrogen receptor α (ERα), progesterone receptor (PR) and c-erbB2 (HER-2/neu) was assessed by immunohistochemistry.</p> <p>Results</p> <p>Histological examination showed 83 out of 310 (26.8%) carcinomas of various subtypes. Immunohistochemistry was carried out in 62/83 carcinomas. Decorin was accumulated in breast tissues with MD and MAMCs independently of the presence of malignancy. In contrast, versican was significantly increased only in carcinomas with MAMCs (median ± SE: 42.0 ± 9.1) and MD (22.5 ± 10.1) as compared to normal breast tissue with MAMCs (14.0 ± 5.8), MD (11.0 ± 4.4) and normal breast tissue without mammographic findings (10.0 ± 2.0). Elevated levels of versican were correlated with higher tumor grade and invasiveness in carcinomas with MD and MAMCs, whereas increased amounts of decorin were associated with <it>in situ </it>carcinomas in MAMCs. Stromal deposition of both proteoglycans was related to higher expression of ERα and PR in tumor cells only in MAMCs.</p> <p>Conclusions</p> <p>The specific accumulation of versican in breast tissue with high MD and MAMCs only in the presence of malignant transformation and its association with the aggressiveness of the tumor suggests its possible use as molecular marker in non-palpable breast carcinomas.</p

Expression and Membrane Topology of Anopheles gambiae Odorant Receptors in Lepidopteran Insect Cells

A lepidopteran insect cell-based expression system has been employed to express three Anopheles gambiae odorant receptors (ORs), OR1 and OR2, which respond to components of human sweat, and OR7, the ortholog of Drosophila's OR83b, the heteromerization partner of all functional ORs in that system. With the aid of epitope tagging and specific antibodies, efficient expression of all ORs was demonstrated and intrinsic properties of the proteins were revealed. Moreover, analysis of the orientation of OR1 and OR2 on the cellular plasma membrane through the use of a novel ‘topology screen’ assay and FACS analysis demonstrates that, as was recently reported for the ORs in Drosophila melanogaster, mosquito ORs also have a topology different than their mammalian counterparts with their N-terminal ends located in the cytoplasm and their C-terminal ends facing outside the cell. These results set the stage for the production of mosquito ORs in quantities that should permit their detailed biochemical and structural characterization and the exploration of their functional properties

Detection of HSV-1 and HSV-2 antibodies by enzyme linked immunosorbent assay

Infections with herpes simplex virus are spread worldwide. Labial herpes is caused predominately by HSV-1 type while genital track infections are caused by HSV-2 type and are sexually transmitted. The prevalence of infections with HSV-2 has been difficult to ascertain because of the limitations of the serological assays to differentiate HSV-2 antibodies. The aim of this study was the detection of IgG antibodies against HSV-1 and HSV-2 and the epidemiologic evaluation of infections caused by each type with respect to age and sex. For the purpose of the present study 1,400 serum samples were examined using ELISA technique. Serum samples from individuals 1 to 60 years of age of both sexes were equally distributed in age groups of 5 years period. Children from 0-5 and 6-12 months of age were grouped separately. Serum samples were screened for HSV-1 and HSV-2 antibodies with a commercially available ELISA technique which uses type specific antigens. The analysis of the results of the present study are summarized as follows: A relatively large percentage (70,86%) of Greek population have antibodies against HSV-1 while antibodies against HSV-2 are detected in small percentage (2.64%). Infections with HSV-1 occur during the infancy (25-62%) and gradually increase with age reaching 95% for the age group of 31-35 years old. For both sexes antibodies against HSV-2 were detected in persons greater than 21 years old with the exception of a child (12 months old) that could be due to perinatal transmission delivery. Women showed higher percentage of seropositivity than men for HSV-2 (3,86% for women, 1,43% for men). The percentage for women of the age group of 16-40 years old was found to be 7,2% A value that has to be taken into account for pregnant women because of the danger of perinatal transmission.Σκοπός αυτής της εργασίας υπήρξε η μελέτη της επιδημιολογίας των δύο τύπων του ιού του απλού έρπητα HSV-1 και HSV-2 και η συχνότητα των μολύνσεων για τους οποίους ευθύνεται ο κάθε τύπος χωριστά. Υλικό για αυτή την εργασία απετέλεσαν 1.400 δείγματα ορών με ισάριθμη κατά φύλο και ομάδα ηλικίας κατανομή, ώστε όλες οι πενταετίες από 1-60 ετών καθώς και οι ομάδες ηλικίας από 0-5 και 6-12 μηνών να αντιπροσωπεύονται από 50 δείγματα ορών έκαστη για κάθε φύλο. Για τον προσδιορισμό ειδικών έναντι του κάθε τύπου του ιού αντισωμάτων χρησιμοποιήθηκε η ανοσοενζυμική μέθοδος (ELISA) με αντιδραστήρια του οίκου Ismunit, τα οποία περιλαμβάνουν ειδικά για τον κάθε τύπο του ιού αντιγόνα. Τα αποτελέσματα αυτής της εργασίας μπορούν να συνοψιστούν στα παρακάτω συμπεράσματα: Ένα μεγάλο μέρος (70,86%) του πληθυσμού της Χώρας μας έχει αντισώματα έναντι του HSV-1 ενώ αντισώματα έναντι του HSV-2 διαπιστώνονται μόνο σε μικρή αναλογία (2,64%). Οι περισσότερες μολύνσεις με τον ιό του απλού έρπητα τύπου 1 (HSV-1) πραγματοποιούνται κατά την παιδική ηλικία, όπως προκύπτει από την υψηλή συχνότητα αντισωμάτων (25-62%) σε αυτή την ηλικία. Η συχνότητα αυτή συνεχίζει να αυξάνεται σταδιακά με την πάροδο της ηλικίας με βραδύτερο ρυθμό και προσεγγίζει το 95% σε άτομα ηλικίας 31-35 ετών. Με εξαίρεση ενός βρέφους ηλικίας 12 μηνών το οποίο πιθανόν να μολύνθηκε κατά την περιγεννητική περίοδο, αντισώματα έναντι του HSV-2 διαπιστώθηκαν μόνο σε άτομα μεγαλύτερα των 21 ετών και για τα δύο φύλα. Οι γυναίκες μολύνονται με τον HSV-2 σε μεγαλύτερη αναλογία από τους άνδρες (3,86% έναντι 1,43%). Η μέση συχνότητα μολύνσεων με τον HSV-2 σε γυναίκες ηλικίας 16-40 ετών ανέρχεται σε 7,2% γεγονός το οποίο επισημαίνει τον κίνδυνο περιγεννητικής μόλυνσης του νεογνού κατά και μετά τον τοκετό

Serglycin inhibits the classical and lectin pathways of complement via its glycosaminoglycan chains: Implications for multiple myeloma

Serglycin (SG) is a proteoglycan expressed by hematopoietic cells and is constitutively secreted by multiple myeloma (MM) cells. SG participates in the regulation of various inflammatory events. We found that SG secreted by human MM cell lines inhibits both the classical and lectin pathways of complement, without influencing alternative pathway activity. The inhibitory effect of SG is due to direct interactions with C1q and mannose-binding lectin (MBL). C1q-binding is mediated through the glycosaminoglycan moieties of SG, whereas MBL binds additionally to SG protein core. Interactions between SG and C1q as well as MBL are diminished in the presence of chondroitin sulfate type E. In addition, we localized the SG-binding site to the collagen-like stalk of C1q. Interactions between SG and C1q as well as MBL are ionic in character and only the interaction with MBL was found to be partially dependent on the presence of calcium. We found the serum levels of SG to be elevated in patients with MM compared to healthy controls. Moreover, we found that SG expressed from myeloma plasma cells protects these cells from complement activation induced by treatment with anti-thymocyte immunoglobulins. This might protect myeloma cells during immunotherapy and promote survival of malignant cells

Expression of Syndecan-4 and Correlation with Metastatic Potential in Testicular Germ Cell Tumours

Although syndecan-4 is implicated in cancer progression, there is no information for its role in testicular germ cell tumours (TGCTs). Thus, we examined the expression of syndecan-4 in patients with TGCTs and its correlation with the clinicopathological findings. Immunohistochemical staining in 71 tissue specimens and mRNA analysis revealed significant overexpression of syndecan-4 in TGCTs. In seminomas, high percentage of tumour cells exhibited membranous and/or cytoplasmic staining for syndecan-4 in all cases. Stromal staining for syndecan-4 was found in seminomas and it was associated with nodal metastasis (), vascular/lymphatic invasion (), and disease stage (). Reduced tumour cell associated staining for syndecan-4 was observed in nonseminomatous germ cell tumours (NSGCTs) compared to seminomas. This loss of syndecan-4 was associated with nodal metastasis (), vascular/lymphatic invasion (), and disease stage (). Stromal staining for syndecan-4 in NSGCTs did not correlate with any of the clinicopathological variables. The stromal expression of syndecan-4 in TGCTs was correlated with microvessel density (). Our results indicate that syndecan-4 is differentially expressed in seminomas and NSGCTs and might be a useful marker. Stromal staining in seminomas and reduced levels of syndecan-4 in tumour cells in NSGCTs are related to metastatic potential, whereas stromal staining in TGCTs is associated with neovascularization