Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

Multivalency of targeting ligands provides significantly increased binding strength towards their molecular targets. Here, we report the development of a novel heptameric targeting system, with general applications, constructed by fusing a target-binding domain with the heptamerization domain of the Archaeal RNA binding protein Sm1 through a flexible hinge peptide. The previously reported affibody molecules against EGFR and HER2, ZEGFR and ZHER2, were used as target binding moieties. The fusion molecules were highly expressed in E. coli as soluble proteins and efficiently self-assembled into multimeric targeting ligands with the heptamer as the predominant form. We demonstrated that the heptameric molecules were resistant to protease-mediated digestion or heat- and SDS-induced denaturation. Surface plasmon resonance (SPR) analysis showed that both heptameric ZEGFR and ZHER2 ligands have a significantly enhanced binding strength to their target receptors with a nearly 100 to 1000 fold increase relative to the monomeric ligands. Cellular binding assays showed that heptameric ligands maintained their target-binding specificities similar to the monomeric forms towards their respective receptor. The non-toxic property of each heptameric ligand was demonstrated by the cell proliferation assay. In general,, the heptamerization strategy we describe here could be applied to the facile and efficient engineering of other protein domain- or short peptide-based affinity molecules to acquire significantly improved target-binding strengths with potential applications in the targeted delivery of various imaging or therapeutic agents.

Developmental Expression of Secretory β-1,4-endoglucanases in the Subventral Esophageal Glands of Heterodera glycines

Two β-1,4-endoglucanases (EGases), Hg-eng-1 and Hg-eng-2, were recently cloned from the soybean cyst nematode, Heterodera glycines, and their expression was shown in the subventral esophageal glands of hatched second-stage juveniles (J2). We examined the expression of these EGases in the subventral glands of all post-embryonic life stages of H. glycines by in situ hybridization and immunolocalization. The first detectable accumulation of EGase mRNAs occurred in the subventral glands of unhatched J2. EGase transcripts remained detectable in J2 after hatching and during subsequent root invasion. However, in late parasitic J2 and third-stage juveniles (J3), the percentage of individuals that showed EGase transcripts decreased. In female fourth-stage juveniles and adult females, EGase transcripts were no longer detected in the subventral glands. EGase hybridization signal reappeared in unhatched males coiled within the J3 cuticle, and transcripts were also present in the subventral glands of migratory adult males. Immunofluorescence labeling showed that EGase translation products are most abundantly present in the subventral glands of preparasitic J2, migratory parasitic J2, and adult males. The presence of EGases predominantly in the migratory stages suggests that the enzymes are used by the nematodes to soften the walls of root cells during penetration and intracellular migration

Uber den Einfluss der Phrenicusexairese auf die Magenfunktion. II. Mitteilung : Experimentelle Studien an Hunden mit Hilfe des Rontgenverfahren

Was den Einfluss der Phrenicusexairese auf die Magenfunktion, besonders Magenfunktionsveranderung direkt nach Phrenicusexairese mit Hilfe des Magenkurvenverfahrens anbelangt, hat Verfasser schon in der vorigen Mitteilung berichtet. Er hat weiternoch uber ihre Dauerresultate geforscht. Zur Untersuchung hat er das Rontgenverfahren angewandt, womit er die Bewegungs- und Passagezustande des Inhaltes beobachten konnte. Als Versuchstier hat er erwachsene kleine Hunde gebraucht. Die Resultate sind folgendermassen:- 1. Der Magen verandert seine Form durch den Hochstand infolge gelahmten Zwerchfells, d. h. bei der rechtseitigen Phrenicusexairese wird Pylorus nach oben gezogen und bei der linksseitigen nach links unten. 2. Der Magentonus sinkt deutlich bald nach der Operation ab und zeigt den Zustand von Dilatation und Atonie. Und er stellt sich allmahlich wieder her bis zur Norm und zwar bei einseitiger Exairese nach einigen Wochen, bei beiderseitiger nach einigen Monaten. 3. Die Entleerung des Mageninhalts wird gestort und es verzogert sich der Initialzeitpunkt, in dem der Inhalt den Pylorus passiert, und auch die vollige Entleerungszeit. Diese Storungen zeigen eine allmahliche Wiederherstellung und sie kommen mit der Peristaltik zum normalen erst nach einigen Wochen zuruck. 4. Auch zeigt das Duodenum die Herabsetzung des Tonus und Dilatatation und der Dunndarm zeigt schnelle Passage und schmale Rontgenbilder. (Autoreferat.

Prolonged Cadmium Exposure Alters Migration Dynamics and Increases Heterogeneity of Human Uterine Fibroid Cells—Insights from Time Lapse Analysis

Cadmium (Cd) is one of the most prevalent environmental heavy metal contaminants and is considered an endocrine disruptor and carcinogen. In women with uterine fibroids, there is a correlation between blood Cd levels and fibroid tumor size. In this study, fibroid cells were exposed to 10 µM CdCl2 for 6 months and a fast-growing Cd-Resistant Leiomyoma culture, termed CR-LM6, was recovered. To characterize the morphological and mechanodynamic features of uterine fibroid cells associated with prolonged Cd exposure, we conducted time lapse imaging using a Zeiss confocal microscope and analyzed data by Imaris and RStudio. Our experiments recorded more than 64,000 trackable nuclear surface objects, with each having multiple parameters such as nuclear size and shape, speed, location, orientation, track length, and track straightness. Quantitative analysis revealed that prolonged Cd exposure significantly altered cell migration behavior, such as increased track length and reduced track straightness. Cd exposure also significantly increased the heterogeneity in nuclear size. Additionally, Cd significantly increased the median and variance of instantaneous speed, indicating that Cd exposure results in higher speed and greater variation in motility. Profiling of mRNA by NanoString analysis and Ingenuity Pathway Analysis (IPA) strongly suggested that the direction of gene expression changes due to Cd exposure enhanced cell movement and invasion. The altered expression of extracellular matrix (ECM) genes such as collagens, matrix metallopeptidases (MMPs), secreted phosphoprotein 1 (SPP1), which are important for migration contact guidance, may be responsible for the greater heterogeneity. The significantly increased heterogeneity of nuclear size, speed, and altered migration patterns may be a prerequisite for fibroid cells to attain characteristics favorable for cancer progression, invasion, and metastasis

Bulletin mensuel de statistique / Institut national de la statistique et des études économiques...

septembre 19941994/09 (N9)

Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

Multivalency of targeting ligands provides significantly increased binding strength towards their molecular targets. Here, we report the development of a novel heptameric targeting system, with general applications, constructed by fusing a target-binding domain with the heptamerization domain of the Archaeal RNA binding protein Sm1 through a flexible hinge peptide. The previously reported affibody molecules against EGFR and HER2, Z(EGFR) and Z(HER2), were used as target binding moieties. The fusion molecules were highly expressed in E. coli as soluble proteins and efficiently self-assembled into multimeric targeting ligands with the heptamer as the predominant form. We demonstrated that the heptameric molecules were resistant to protease-mediated digestion or heat- and SDS-induced denaturation. Surface plasmon resonance (SPR) analysis showed that both heptameric Z(EGFR) and Z(HER2) ligands have a significantly enhanced binding strength to their target receptors with a nearly 100 to 1000 fold increase relative to the monomeric ligands. Cellular binding assays showed that heptameric ligands maintained their target-binding specificities similar to the monomeric forms towards their respective receptor. The non-toxic property of each heptameric ligand was demonstrated by the cell proliferation assay. In general,, the heptamerization strategy we describe here could be applied to the facile and efficient engineering of other protein domain- or short peptide-based affinity molecules to acquire significantly improved target-binding strengths with potential applications in the targeted delivery of various imaging or therapeutic agents.

Genistein: Dual Role in Women’s Health

Advanced research in recent years has revealed the important role of nutrients in the protection of women’s health and in the prevention of women’s diseases. Genistein is a phytoestrogen that belongs to a class of compounds known as isoflavones, which structurally resemble endogenous estrogen. Genistein is most often consumed by humans via soybeans or soya products and is, as an auxiliary medicinal, used to treat women’s diseases. In this review, we focused on analyzing the geographic distribution of soybean and soya product consumption, global serum concentrations of genistein, and its metabolism and bioactivity. We also explored genistein’s dual effects in women’s health through gathering, evaluating, and summarizing evidence from current in vivo and in vitro studies, clinical observations, and epidemiological surveys. The dose-dependent effects of genistein, especially when considering its metabolites and factors that vary by individuals, indicate that consumption of genistein may contribute to beneficial effects in women’s health and disease prevention and treatment. However, consumption and exposure levels are nuanced because adverse effects have been observed at lower concentrations in in vitro models. Therefore, this points to the duplicity of genistein as a possible therapeutic agent in some instances and as an endocrine disruptor in others