Inhibin/activin subunits alpha, beta-A and beta-B are differentially expressed in normal human endometrium throughout the menstrual cycle

Inhibins are dimeric glycoproteins composed of an alpha (alpha) subunit and one of two possible beta (beta-) subunits (betaA or betaB). The aims of this study were to assess the frequency and tissue distribution patterns of the inhibin subunits in normal human endometrium. Samples from human endometrium from proliferative phase (PP; n=32), early secretory phase (ES; n=10) and late secretory phase (LS; n=12) were obtained. Immunohistochemistry, immunofluorescence and a statistical analysis were performed. All three inhibin subunits were expressed by normal endometrium by immunohistochemistry and immunofluorescence. Inhibin-alpha was primarily detected in glandular epithelial cells, while inhibin-beta subunits were additionally localised in stromal tissue. Inhibin-alpha staining reaction increased significantly between PP and ES (P<0.05), PP and LS (P<0.01), and ES and LS (P<0.02). Inhibin-betaA and -betaB were significant higher in LS than PP (P<0.05) and LS than ES (P<0.05). All three inhibin subunits were expressed by human endometrium varying across the menstrual cycle. This suggests substantial functions in human implantation of inhibin-alpha subunit, while stromal expression of the beta subunits could be important in the paracrine signalling for adequate endometrial maturation. The distinct expression in human endometrial tissue suggests a synthesis of inhibins into the lumen and a predominant secretion of activins into the stroma.     

An investigation on the expression of miRNAs including miR-144 and miR-34a in plasma samples of RET-positive and RET-negative medullar thyroid carcinoma patients

Medullary thyroid carcinoma (MTC) is a scarce cancerous disease, originating from parafollicular C cells of the thyroid gland. MTC can be manifested as an aggressive carcinoma with metastasis, especially in sporadic forms. Mutations of the rearranged during transfection (RET) proto-oncogene occurs in all hereditary and a few somatic MTCs, so detection of RET mutations is needed for prompt and appropriate treatment. MicroRNAs (miRNAs) are noncoding regulatory RNAs. Extensive studies have done in progress or suppression of several types of cancers such as MTCs with the remarkable application as prognostic markers. Of the effective miRNAs in cancers, miR-144 and miR-34 were evaluated in our study. Blood samples of 25 RET-positive and 25 RET-negative blood samples of patients with MTC were evaluated for these miRNAs, using quantitative real-time polymerase chain reaction (RT-qPCR). Analysis of the results was performed by the 2 ^−ΔΔCt method, showing that miR-144 and miR-34a expression had a relative increase in patients with MTC compared with normal control samples and also in RET positives versus RET negatives. We recruited 50 out of 350 MTC plasma samples (27 female and 23 male) which were selected based on RET mutation in exon 11 (25 RET-positive and 25 RET-negative), with a mean ± SD age of 37.04 ± 1.74 years. Receiver operating characteristic (ROC) curve analysis was done to investigate the prognostic value of these miRNAs; although, they showed no significant prognostic value as MTC biomarkers in plasma samples. In conclusion, miRNAs can be used as biomarkers of cancers such as MTC; however, more studies are needed to find the best candidate miRNAs for the diagnosis of cancers.     

Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression

N‐Glycans of human proteins possess both α2,6‐ and α2,3‐linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3‐linkage due to the absence of α2,6‐sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)‐producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC‐Sambucus nigra (SNA) lectin that preferentially binds α2,6‐linked SA. The presence of α2,6‐linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2‐fold compared to the control. For host cell engineering, the CHOZN^® GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single‐cell clones were derived from the enriched population and selected based on FITC‐SNA staining and St6gal1 expression. Two clones (“ST6GAL1 OE Clone 31 and 32”) were confirmed for the presence of α2,6‐linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6‐linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human‐like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of “bio‐better” protein therapeutics and cell culture vaccine production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:334–346, 2015     

Increasing the expression of programmed death ligand 2 (PD-L2) but not 4-1BB ligand in colorectal cancer cells

Molecular Biology Reports - Immune checkpoint (ICP) molecules modulate the immune response by either inducing or preventing T cell activation. Over-expression of some ICPs on malignant cells has...     

Fabrication of Au/Fe3O4/RGO based aptasensor for measurement of miRNA‐128, a biomarker for acute lymphoblastic leukemia (ALL)

Due to their high sensitivity, simplicity, portability, self‐contained, and low cost, the development of electrochemical biosensors is a beneficial way to diagnose and anticipate many types of cancers. An electrochemical nanocomposite‐based aptasensor is fabricated for the determination of miRNA‐128 concentration as the acute lymphoblastic leukemia (ALL) biomarker for the first time. The aptamer chains were immobilized on the surface of the glassy carbon electrode (GCE) through gold nanoparticles/magnetite/reduced graphene oxide (AuNPs/Fe₃O₄/RGO). Fast Fourier transform infrared (FTIR), X‐ray diffraction (XRD), vibrating sample magnetometer (VSM), and transmission electron microscopy (TEM) were used to characterize synthesized nanomaterials. Cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS) were used to characterize the modified GCE in both label‐free and labeled methods. The results indicate that the modified working electrode has high selectivity and for miRNA‐128 over other biomolecules. The hexacyanoferrate redox system typically operated at around 0.3 V (vs. Ag/AgCl), and the methylene blue redox system ran at about 0 V, were used as an electrochemical probe. The detection limit and linear detection range for hexacyanoferrate and methylene blue are 0.05346 fM, 0.1–0.9 fM, and 0.005483 fM, 0.01–0.09 fM, respectively. The stability and diffusion control analyses were performed as well. In both label‐free and labeled methods, the modified electron showed high selectivity for miRNA‐128. The use of methylene blue as a safer redox mediator caused miRNA‐128 to be detected with greater accuracy at low potentials in PBS media. The findings also show the substantial improvement in detection limit and linearity by using reduced graphene oxide‐magnetite‐gold nanoparticles that can be verified by comparing with previous studies on the detection of other miRNAs.     

Formation of ethyl β-xylopyranoside during simultaneous saccharification and co-fermentation of paper sludge

An unexpected product was detected during simultaneous saccharification and co-fermentation (SSCF) of paper sludge using added commercial cellulase (Spezyme CP) by Saccharomyces cerevisiae RWB222, S. cerevisiae D5A, and Zymomonas mobilis 8b. Based on glycosyl composition analysis, linkage analysis and NMR analysis, the compound was identified as ethyl β-xylopyranoside (EXP). The carbon mass balance analysis showed up to 25% of xylan originally present in paper sludge was converted to EXP. EXP formation was found in simultaneous saccharification of beech wood xylan as well, and later proved to be produced by the Trichoderma reesei derived cellulase and hemicellulase mixture (Spezyme CP) during the course of xylan hydrolysis in the presence of ethanol, and its production increased with an increased concentration of ethanol, xylan, and T. reesei enzyme. Similar condensation reactions were also observed with other alcohols. These alcoholysis reactions were found to be reversible. Thermoanaerobacterium saccharolyticum was found to be able to degrade EXP.     

Acidity enhances the effectiveness of active chemical defensive secretions of sea hares, <Emphasis Type="Italic">Aplysia californica</Emphasis>, against spiny lobsters, <Emphasis Type="Italic">Panulirus interruptus</Emphasis>

Sea hares such as Aplysia californica, gastropod molluscs lacking a protective shell, can release a purple cloud of chemicals when vigorously attacked by predators. This active chemical defense is composed of two glandular secretions, ink and opaline, both of which contain an array of compounds. This secretion defends sea hares against predators such as California spiny lobsters Panulirus interruptus via multiple mechanisms, one of which is phagomimicry, in which secretions containing feeding chemicals attract and distract predators toward the secretion and away from the sea hare. We show here that ink and opaline are highly acidic, both having a pH of ∼5. We examined if the acidity of ink and opaline affects their phagomimetic properties. We tested behavioral and electrophysiological responses of chemoreceptor neurons in the olfactory and gustatory organs of P. interruptus, to ink and opaline of A. californica within their natural range of pH values, from ∼5 to 8. Both behavioral and electrophysiological responses to ink and opaline were enhanced at low pH, and low pH alone accounted for most of this effect. Our data suggest that acidity enhances the phagomimetic chemical defense of sea hares.