Introduction of hematoxylin as an electroactive label for DNA biosensors and its employment in detection of target DNA sequence and single-base mismatch in human papilloma virus corresponding to oligonucleotide

For the detection of DNA hybridization, a new electrochemical biosensor was developed on the basis of the interaction of hematoxylin with 20-mer deoxyoligonucleotides (from human papilloma virus, HPV). The study was performed based on the interaction of hematoxylin with an alkanethiol DNA probe self-assembled gold electrode (ss-DNA/AuE) and its hybridization form (ds-DNA/AuE). The optimum conditions were found for the immobilization of HPV probe on the gold electrode (AuE) surface and its hybridization with the target DNA. Electrochemical detection of the self-assembled DNA and the hybridization process were performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range where the accumulated hematoxylin at the modified electrode was electroactive. Observing a remarkable difference between the voltammetric signals of the hematoxylin obtained from different hybridization samples (non-complementary, mismatch and complementary DNAs), we confirmed the potential of the developed biosensor in detecting and discriminating the target complementary DNA from non-complementary and mismatch oligonucleotides. Under optimum conditions, the electrochemical signal had a linear relationship with the concentration of the target DNA ranging from 12.5 nM to 350.0 nM, and the detection limit was 3.8 nM.     

Structural stabilization and functional improvement of horseradish peroxidase upon modification of accessible lysines: experiments and simulation

Horseradish peroxidase (HRP) is an important heme enzyme with enormous medical diagnostic, biosensing, and biotechnological applications. Thus, any improvement in the applicability and stability of the enzyme is potentially interesting. We previously reported that covalent attachment of an electron relay (anthraquinone 2-carboxylic acid) to the surface-exposed Lys residues successfully improves electron transfer properties of HRP. Here we investigated structural and functional consequences of this modification, which alters three accessible charged lysines (Lys-174, Lys-232, and Lys-241) to the hydrophobic anthraquinolysine residues. Thermal denaturation and thermoinactivation studies demonstrated that this kind of modification enhances the conformational and operational stability of HRP. The melting temperature increased 3 degrees C and the catalytic efficiency enhanced by 80%. Fluorescence and circular dichroism investigations suggest that the modified HRP benefits from enhanced aromatic packing and more buried hydrophobic patches as compared to the native one. Molecular dynamics simulations showed that modification improves the accessibility of His-42 and the heme prosthetic group to the peroxide and aromatic substrates, respectively. Additionally, the hydrophobic patch, which functions as a binding site or trap for reducing aromatic substrates, is more extended in the modified enzyme. In summary, this modification produces a new derivative of HRP with enhanced electron transfer properties, catalytic efficiency, and stability for biotechnological applications.     

The effect of Ccnb1ip1 insulator on monoclonal antibody expression in Chinese hamster ovary cells

Background

The increasing need for therapeutic monoclonal antibodies (mAbs) entails the development of innovative and improved expression strategies. Chromatin insulators have been utilized for the enhancement of the heterologous proteins in mammalian cells.

Methods and results

In the current study the Ccnb1ip1 gene insulator element was utilized to construct a novel vector system for the expression of an anti-CD52 mAb in Chinese hamster ovary (CHO) cells. The insulator containing (pIns-mAb) and control (pmAb) vectors were generated and stable cell pools were established using these constructs. The expression level in the cells created with pIns-mAb vector was calculated to be 233 ng/mL, and the expression rate in the control vector was 210 ng/mL, which indicated a 10.9% increase in mAb expression in pIns-mAb pool. In addition, analysis of mAb expression in clonal cells established from each pool showed a 10% increase in antibody productivity in the highest mAb producing clone derived from the pIns-mAb pool compared to the clone isolated from pmAb pool.

Conclusions

More studies are needed to fully elucidate the effects of Ccnb1ip1 gene insulator on recombinant therapeutic protein expression in mammalian cells. The combination of this element with other chromatin-modifying elements might improve its augmentation effect which could pave the way for efficient and cost-effective production of therapeutic drugs.

     

MicroRNAs in cancer drug resistance: Basic evidence and clinical applications

Development of drug resistance has considerably limited the efficacy of cancer treatments, including chemotherapy and targeted therapies. Hence, understanding the molecular mechanisms underpinning the innate or the acquired resistance to these therapies is critical to improve drug efficiency and clinical outcomes. Several studies have implicated microRNAs (miRNA) in this process. MiRNAs repress gene expression by specific binding to complementary sequences in the 3' region of target messenger RNAs (mRNAs), followed by target mRNA degradation or blocked translation. By targeting molecules specific to a particular pathway within tumor cells, the new generation of cancer treatment strategies has shown significant advantages over conventional chemotherapy. However, the long-term efficacy of targeted therapies often remains poor, because tumor cells develop resistance to such therapeutics. Targeted therapies often involve monoclonal antibodies (mAbs), such as those blocking the ErB/HER tyrosine kinases, epidermal growth factor receptor (cetuximab) and HER2 (trastuzumab), and those inhibiting vascular endothelial growth factor receptor signaling (e.g., bevacizumab). Even though these are among the most used agents in tumor medicine, clinical response to these drugs is reduced due to the emergence of drug resistance as a result of toxic effects in the tumor microenvironment. Research on different types of human cancers has revealed that aberrant expression of miRNAs promotes resistance to the aforementioned drugs. In this study, we review the mechanisms of tumor cell resistance to mAb therapies and the role of miRNAs therein. Emerging treatment strategies combine therapies using innovative miRNA mimics or antagonizers with conventional approaches to maximize outcomes of patients with cancer.     

Conformation‐specific antibodies against multiple amyloid protofibril species from a single amyloid immunogen

We engineered and employed a chaperone‐like amyloid‐binding protein Nucleobindin 1 (NUCB1) to stabilize human islet amyloid polypeptide (hIAPP) protofibrils for use as immunogen in mice. We obtained multiple monoclonal antibody (mAb) clones that were reactive against hIAPP protofibrils. A secondary screen was carried out to identify clones that cross‐reacted with amyloid beta‐peptide (Aβ42) protofibrils, but not with Aβ40 monomers. These mAbs were further characterized in several in vitro assays, in immunohistological studies of a mouse model of Alzheimer's disease (AD) and in AD patient brain tissue. We show that mAbs obtained by immunizing mice with the NUCB1‐hIAPP complex cross‐react with Aβ42, specifically targeting protofibrils and inhibiting their further aggregation. In line with conformation‐specific binding, the mAbs appear to react with an intracellular antigen in diseased tissue, but not with amyloid plaques. We hypothesize that the mAbs we describe here recognize a secondary or quaternary structural epitope that is common to multiple amyloid protofibrils. In summary, we report a method to create mAbs that are conformation‐sensitive and sequence‐independent and can target more than one type of protofibril species.     

Factors affecting secondary sex ratio in Iranian Holsteins

The objective of this study was to evaluate the factors affecting secondary sex ratio (SSR) in Iranian Holsteins. Data of 942,941 Holstein calving events from the Animal Breeding Center of Iran, recorded between January 1996 and December 2007, were used in the analysis. A multivariable logistic regression model was used to model the logit of the probability of a male calf being born. Male births accounted for 49.6% of the total observations. The ratio of males to females varied from 52.5:47.5 in calving year 1996-1999 (odds ratio (OR) = 1.18; P < 0.0001), to 48.5:51.5 in calving year 2004-2007. The greatest occurrence of male births was observed in spring (OR = 1.02; P < 0.0001), and the lowest incidence of male births was for summer or fall calvings. Also, the frequency of male births decreased from parity 1 to parity 4 and beyond (P < 0.0001; OR = 1.11). The greatest number of sires had the SSR equal to 0.5 with a minimum SSR of 32% while the maximum was 97%. Among cows that had a male birth, the chance of delivering a male calf again was 25.5% when cows had delivered a male once (OR = 1.14; P < 0.0001), and 12.7% if a male calf was delivered twice by a cow. This indicated that characteristics peculiar to the dam influence the sex of her offspring and suggests some degree of repeatability of calf sex within cows.     

Plasmodium falciparum: sequence analysis of the gene encoding the C-terminus region of the merozoite surface protein-1, a potential malaria vaccine antigen, in Iranian clinical isolates

C-terminal region of merozoite surface protein-1 of Plasmodium falciparum (PfMSP-1) isolated from different parts of the world revealed sequence variability, however no data exist on sequence heterogeneity of this region from Iran. To address this question, DNA encoding the carboxyl (C)-terminal region of PfMSP-1 was amplified in 144 Iranian P. falciparum clinical isolates, using allele type-specific primers. In this study both MAD20 (88.2%) and K1 (7.6%) types were detected. Sequence analysis of 33 and 92 fragments corresponding to pfmsp-1(42) and pfmsp-1(19) revealed eight (15MAD1-15MAD7 and 15KCH) and five [A1 (E/TSR/L), A2 (Q/KNG/F), A3 (E/KNG/F), A4 (E/TSG/L), and A5 (Q/KNG/L)] distinct haplotypes, respectively. E/TSG/L variant type was the predominant haplotype, and reported only from Thailand and India, but E/KNG/L is widespread in Africa, Asia, and Latin America; but not found among Iranian isolates. In summary, result of this study indicates limited antigenic diversity, and thus support the potential utility of the C-terminal region of PfMSP-1 in designing polyvalent vaccine constructs.