The Use of Sensitive Chemical Antibodies for Diagnosis: Detection of Low Levels of Epcam in Breast Cancer

EpCAM is expressed at low levels in a variety of normal human epithelial tissues, but is overexpressed in 70–90% of carcinomas. From a clinico-pathological point of view, this has both prognostic and therapeutic significance. EpCAM was first suggested as a therapeutic target for the treatment of epithelial cancers in the 1990s. However, following several immunotherapy trials, the results have been mixed. It has been suggested that this is due, at least in part, to an unknown level of EpCAM expression in the tumors being targeted. Thus, selection of patients who would benefit from EpCAM immunotherapy by determining EpCAM status in the tumor biopsies is currently undergoing vigorous evaluation. However, current EpCAM antibodies are not robust enough to be able to detect EpCAM expression in all pathological tissues. Here we report a newly developed EpCAM RNA aptamer, also known as a chemical antibody, which is not only specific but also more sensitive than current antibodies for the detection of EpCAM in formalin-fixed paraffin-embedded primary breast cancers. This new aptamer, together with our previously described aptamer, showed no non-specific staining or cross-reactivity with tissues that do not express EpCAM. They were able to reliably detect target proteins in breast cancer xenograft where an anti-EpCAM antibody (323/A3) showed limited or no reactivity. Our results demonstrated a more robust detection of EpCAM using RNA aptamers over antibodies in clinical samples with chromogenic staining. This shows the potential of aptamers in the future of histopathological diagnosis and as a tool to guide targeted immunotherapy.     

Protection of Shrimp Penaeus monodon from WSSV Infection Using Antisense Constructs

White spot syndrome caused by white spot syndrome virus (WSSV) is one of the most threatening diseases of shrimp culture industry. Previous studies have successfully demonstrated the use of DNA- and RNA-based vaccines to protect WSSV infection in shrimp. In the present study, we have explored the protective efficacy of antisense constructs directed against WSSV proteins, VP24, and VP28, thymidylate synthase (TS), and ribonucleotide reductase-2 (RR2) under the control of endogenous shrimp histone-3 (H3) or penaedin (Pn) promoter. Several antisense constructs were generated by inserting VP24 (pH3–VP24, pPn–VP24), VP28 (pH3–VP28, pPn–VP28), TS (pH3–TS, pPn–TS), and RR2 (pH3–RR2) in antisense orientation. These constructs were tested for their protective potential in WSSV infected cell cultures, and their effect on reduction of the viral load was assessed. A robust reduction in WSSV copy number was observed upon transfection of antisense constructs in hemocyte cultures derived from Penaeus monodon and Scylla serrata. When tested in vivo, antisense constructs offered a strong protection in WSSV challenged P. monodon. Constructs expressing antisense VP24 and VP28 provided the best protection (up to 90 % survivability) with a corresponding decrease in the viral load. Our work demonstrates that shrimp treated with antisense constructs present an efficient control strategy for combating WSSV infection in shrimp aquaculture.     

Balanced Carrier Mobilities: Not a Necessary Condition for High‐Efficiency Thin Organic Solar Cells as Determined by MIS‐CELIV

A novel technique based upon injection‐charge extraction by linearly increasing voltage (i‐CELIV) in a metal‐insulator‐semiconductor (MIS) diode structure is described for studying charge transport in organic semiconductors. The technique (MIS‐CELIV) allows selective measurement of both electron and hole mobilities of organic solar cells with active layers thicknesses representative of operational devices. The method is used to study the model high efficiency bulk heterojunction combination poly[N‐9′′‐heptadecanyl‐2,7‐carbazole‐alt‐5,5‐(4′,7′‐di‐2‐thienyl‐2′,1′,3′‐benzothiadiazole)] (PCDTBT) and [6,6]‐phenyl C70‐butyric acid methyl ester (PC70BM) at various blend ratios. The absence of bipolar transport in PCDTBT‐and‐PC70BM‐only diodes is shown and strongly imbalanced carrier mobility is found in the most efficient “optimized” blend ratios. The mobility measurements are correlated with overall device performance and it is found that balanced and high charge carrier mobility are not necessarily required for high efficiencies in thin film organic solar cells.     

Screening of IR50?×?Rathu Heenati F7 RILs and Identification of SSR Markers Linked to Brown Planthopper (Nilaparvata lugens St?l) Resistance in Rice (Oryza sativa L.)

Brown planthopper (Nilaparvata lugens St?l) is one of the major insect pests of rice. A Sri Lankan indica rice cultivar Rathu Heenati was found to be resistant to all biotypes of the brown planthopper. In the present study, a total of 268 F₇ RILs of IR50 and Rathu Heenati were phenotyped for their level of resistance against BPH by the standard seedbox screening test (SSST) in the greenhouse. A total of 53 SSR primers mapped on the chromosome 3 were used to screen the polymorphism between the parents IR50 and Rathu Heenati, out of which eleven were found to be polymorphic between IR50 and Rathu Heenati. The eleven primers that have shown polymorphism between the IR50 and Rathu Heenati parents were genotyped in a set of five resistant RILs and five susceptible RILs along with the parents for co-segregation analysis. Among the eleven primers, two primers namely RM3180 (18.22 Mb) and RM2453 (20.19 Mb) showed complete co-segregation with resistance. The identification of SSR markers linked with BPH resistant could be used for the maker assisted selection (MAS) program in rice breeding and to map the resistant genes on rice chromosomes for further gene cloning.     

Functional models for catechol dioxygenases: iron(III) complexes of cis-facially coordinating linear 3N ligands

A series of 1:1 iron(III) complexes of simple and sterically hindered tridentate 3N donor ligands have been synthesized and studied as functional models for catechol dioxygenases. All of them are of the type [FeLCl3], where L is bis(pyrid-2-yl-methyl)amine (L1), N,N-bis(benzimidazol-2-ylmethyl)amine (L2), N-methyl-N'-(pyrid-2-ylmethyl)ethylenediamine (L3), N,N-dimethyl-N'-(pyrid-2-ylmethyl)-ethylenediamine (L4) and N-phenyl-N'-(pyrid-2-ylmethyl)ethylenediamine (L5). They have been characterised by spectral and electrochemical methods. The X-ray crystal structure of the complex [Fe(L4)Cl3] has been successfully determined. The complex crystallizes in the triclinic space group P1 with a = 7.250(6), b = 8.284(3), c = 12.409(4) angstroms, alpha = 80.84(3) degrees, beta = 86.76(6) degrees, gamma = 72.09(7) degrees and Z = 2. It possesses a distorted octahedral geometry in which the L4 ligand is cis-facially coordinated to iron(III) and the chloride ions occupy the remaining coordination sites. The systematic variation in the ligand donor atom type significantly influences the Lewis acidity of the iron(III) center and hence the binding interaction of the complexes with simple and substituted catechols. The spectroscopic and electrochemical properties of the catecholate complexes generated in situ have been investigated. All the complexes catalyze mainly the oxidative intradiol cleavage of 3,5-di-tert-butylcatechol (H2DBC) in the presence of dioxygen, which is unexpected of the cis-facial coordination of the ligands. The rate of intradiol catechol cleavage reaction depends upon the Lewis acidity of iron(III) center and steric demand and hydrogen-bonding functionalities of the ligands. Interestingly, the electron-sink property of N-phenyl substituent in [Fe(L5)Cl3] complex leads to enhancement in rate of cleavage. All these observations provide support to the substrate activation mechanism proposed for intradiol-cleaving enzymes.