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Background: The Papanicolaou (Pap) screen has been successful in reducing cervical cancer; but exhibits low sensitivity when detecting cervical dysplasia. Use of molecular biomarkers in Pap tests may improve diagnostic accuracy., Design: Monoclonal antibodies to Minichromosome Maintenance Protein 2 (MCM2) and DNA Topoisomerase II α (TOP2A) were selected for use in IHC based on their ability to differentiate normal from diseased cervical tissues in tissue microarrays. Enhanced Green Fluorescent Protein Western blot analysis was used to help identify binding epitopes specific to MCM2 and TOP2A antibody clones. Antibody affinity was determined by solution phase affinity measurement and immunohistochemistry was performed using high affinity MCM2 or TOP2A antibodies on serial histological sections., Results: Antibody clones to MCM2 and TOP2A clones were selected based on their ability to detect over expression in abnormal cervical epithelia. In IHC, MCM2-27C5.6 and MCM2-26H6.19 demonstrated superior staining in abnormal cervical tissue over the MCM2-CRCT2.1 antibody. A combination of MCM2 and TOP2A antibodies showed greater staining when compared to staining with any of the antibodies alone on serial histological sections. Distinct linear epitopes were elucidated for each of the MCM2 and TOP2A clones. Affinity values (Kd) for MCM2 or TOP2A antibodies had a similar range. In a research study, the MCM2 and TOP2A (BD ProEx™ C) antibody cocktail showed increased epithelia staining with increasing dysplasia. The use of BD ProEx™ C in combination with H&E staining enhanced immunohistochemical discrimination of dysplastic and non-dysplastic FFPE cervical tissue specimens., Conclusions: BD ProEx™ C containing MCM2 and TOP2A antibodies showed strong specific nuclear staining that correlated with increased dysplasia and lesion severity. Enhanced performance of the antibodies was linked to their unique topography recognition. BD ProEx™ C incorporates antibodies that enhance detection of CIN2+ cervical disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The chromosomal translocation t(8;21) often found in acute myeloid leukemia generates an oncogenic fusion protein AML1-ETO. This chimeric oncoprotein disrupts wild-type AML1 function and dysregulates genes important for normal myelopoiesis. Monoclonal antibodies that can capture and detect the AML1-ETO fusion protein would help with early diagnosis and treatment prognosis of acute myeloid leukemia. We report the development of murine monoclonal antibodies (MAbs) that specifically bind epitopes encoded by either AML1 or ETO. Since alignment to the human ETO protein indicated almost 100% homology to the mouse ortholog, a strategy was needed to instruct humoral immunity in mice to focus and respond to self-epitopes. Our strategy to develop capture/detector reagents involved producing MAbs that would bind to epitopes within the non-fused myelopic protein (i.e., either AML1 or ETO). This included a process to select antibodies for their ability to also recognize the translocated chromosomal AML1-ETO fusion protein and to identify complementary capture/detector antibody pairs. Construction of a peptide hapten-carrier complex and use of a rapid immunization protocol resulted in IgM-IgG ETO specific MAbs. These MAbs bound specifically to a recombinant form of AML1-ETO fusion protein expressed in HEK and to an endogenous AML1-ETO form of the fusion protein expressed in Kasumi-1. We report the development of murine hybridoma MAbs derived from immunizations with a peptide "self-epitope." Our findings provide a potential strategy to instruct humoral immunity in mice to focus and respond to self-epitopes. This strategy has been validated with the oncogenic fusion protein AML1-ETO involved in acute myeloid leukemia.

Quality control is paramount to reproducibly achieving oligonucleotide therapeutics and diagnostics of superior value. However, incomplete deprotection of nucleoside reactive groups after the automated chemical synthesis of oligonucleotides would result in diminished antisense activity and in erroneous array analysis of gene expression. Mass spectrometry and capillary electrophoresis are used to detect aborted sequences of oligonucleotides, but not to identify and quantify incompletely deprotected oligonucleotides. To address this problem, monoclonal antibodies (MAbs), ELISA, and dot-blot assays were developed for the specific identification and quantification of the commonly used nucleic acid base- and sugar-protecting groups: benzoyl, isobutyryl, isopropylphenoxyacetyl, and dimethoxytrityl. Each MAb was capable of reproducibly detecting 8-32 pmol of the respectively protected nucleoside in an intact DNA or RNA sample composed of 320-640 nmol of the deprotected nucleoside. In a direct comparison, HPLC nucleoside composition analysis of enzyme-hydrolyzed DNA was limited to the detection of 2-5 nmol of protected nucleoside. Using the MAb dot-blot assay, 5 of 16 commercial DNA products obtained from eight different companies were found to have 1.0-5.2% of the benzoyl and isopropylphenoxyacetyl protecting groups remaining. Thus, MAbs selectively identify and quantify picomoles of remaining protecting groups on antisense therapeutics and oligonucleotide diagnostics.

The purpose of this study was to determine whether immunization with purified outer membrane vesicles (OMV) from Treponema pallidum (T.p. ) could elicit Abs capable of killing this organism. It is well established that the immunization of rabbits or mice with killed T.p. or with recombinant T.p. Ags has failed to generate serum killing activity comparable with that of infection-derived immunity. Because of the small amount of T.p. OMV obtainable, a single mouse was immunized with purified OMV. The mouse anti-OMV serum and infection-derived immune rabbit serum (IRS) were compared by reactivities on two-dimensional T.p. immunoblots and by the T.p. immobilization test, a complement-dependent killing assay. Whereas IRS detected >40 Ags, the anti-OMV serum identified only 6 Ags corresponding to proteins identified previously in the outer membrane. T.p. immobilization testing showed that IRS had a 100% killing titer of 1:44 and a 50% killing titer of 1:662. By comparison, the mouse anti-OMV serum had a significantly greater 100% killing titer of 1:1,408 and a 50% killing titer of 1:16,896. Absorption of the anti-OMV serum to remove Ab against outer membrane-associated lipoproteins did not change the 100% killing titer. Freeze-fracture analysis of T.p. incubated in IRS or anti-OMV serum showed that T.p. rare membrane-spanning outer membrane proteins were aggregated. This is the first demonstration of high-titer killing Abs resulting from immunization with defined T.p. molecules; our study indicates that the targets for these Abs are T. p. rare outer membrane proteins.

Populations of normal and tumorigenic epithelial cells were plated in normal (serum supplemented) and a minimal defined medium. The ability of cells to grow in the minimal defined medium vs. the normal medium was related to their tumorigenic ability. All of the clones that were capable of growth in the minimal defined medium demonstrated tumorigenic ability. None of the normal clones survived in the minimal defined medium. As expected, some of the tumorigenic clones could not grow in the minimal medium. However, at no time did a clone that grew in the minimal defined medium fail to demonstrate tumorigenic ability. This validated that this system can be an animal-free means for selecting tumorigenic epithelial cells out of a mixed population.

Introduction: Biomarkers yield important information for early diagnosis of cervical cancer. However, they are rarely applied for prognosis of cervical cancer in Tanzania, where visual inspection assay with acetic acid or Lugol's iodine and Pap test are being used as the standard screening/ diagnostic methods. Methods: This was a retrospective hospital-based cross-sectional study that was conducted to assess cyclin-dependent kinase inhibitor (p16) and topoisomerase II-alpha (TOP2A) proteins expression among women seeking cervical cancer care at Kilimanjaro Christian Medical Centre, Tanzania between May 1, 2017 and May 10, 2018. Immunohistochemistry technique was used to detect the expressions of p16 and TOP2A proteins from the retrieved formalin-fixed and paraffin-embedded (FFPE) cervical biopsies. Results: A total of 145 patients, with a mean age of 52.1 ± 12.9 years, were included in this study. Upon immunohistochemistry staining, 103 (71.0%) and 90 (62.1%) were p16 and TOP2A positive respectively. There was a strong association between histopathological class and p16/TOP2A expression levels (Fisher's exact test, p<0.001). Moreover, there was a strong positive correlation between p16/TOP2A and cancerous cervical lesions (Spearman's rank correlation coefficients = 0.833 and 0.687, p = 0.006 and 0.005, respectively). The age-adjusted odds ratio for predicting cervical cancer lesions were independently significant for p16/TOP2A biomarkers in FFPE cervical tissues [p16: OR = 1.142 (95% CI: 1.059–1.232, p<0.001) and TOP2A: OR = 1.046 (95% CI: 1.008–1.085, p = 0.015)]. Importantly, the diagnostic performance of p16 was higher than that of TOP2A in the diagnosis of cancerous lesions from non-cancerous cervical lesions (sensitivity: 97.2% versus 77.6%, accuracy: 92.8% versus 87.8%, respectively). Conclusion: Our study has highlighted that over-expression of TOP2A is related to the grade of cervical intraepithelial neoplasia but does not predict prognosis in cervical cancer. Similarly, expression of p16 is related to degree of histological dysplasia and malignancy, suggesting its prognostic and predictive value in the management of cervical cancers. Further bigger studies are needed to validate their applications in the early diagnosis of cervical cancer. [ABSTRACT FROM AUTHOR]

Background: To improve the efficiency of early diagnosis systems for cervical cancer, the use of cellular and viral markers for identifying precancerous lesions with a greater probability to progress to cancer has been proposed. Several cellular proteins and markers of oxidative DNA damage have been suggested as possible biomarkers of cervical carcinogenesis; however, they have not been evaluated together. In this study, we analyzed the expression of the cellular markers p16INK4a, Ki-67, CyclinE1, TOP2A/MCM2, and telomerase, as well as the DNA oxidative damage markers ROS and 8-OHdG. The analyses were performed in liquid-based cervical cytology samples or biopsies with premalignant lesions or cervical cancer diagnosis, with the purpose of selecting a panel of biomarkers that allow the identification of precursor lesions with greater risk of progression to cervical cancer.Methods: We analyzed 1485 liquid-based cytology samples, including 239 non-squamous intraepithelial lesions (NSIL), 901 low-grade squamous intraepithelial lesions (LSIL), 54 high-grade squamous intraepithelial lesions (HSIL), and 291 cervical cancers (CC). The biomarkers were analyzed by immunocytochemistry and Human Papilloma Virus (HPV) genotyping with the INNO-LiPA genotyping Extra kit.Results: We found that all tested cellular biomarkers were overexpressed in samples with high risk-HPV infection, and the expression levels increased with the severity of the lesion. TOP2A/MCM2 was the best biomarker for discriminating between LSIL and HSIL, followed by p16INK4a and cyclinE1. Statistical analysis showed that TOP2A/MCM2 provided the largest explanation of HSIL and CC cases (93.8%), followed by p16INK4a (91%), cyclin E1 (91%), Ki-67 (89.3%), and telomerase (88.9%).Conclusions: We propose that the detection of TOP2A/MCM2, p16INK4a and cyclin E1 expression levels is useful as a panel of biomarkers that allow identification of cervical lesions with a higher risk for progression to CC with high sensitivity and precision; this can be done inexpensively, in a single and non-invasive liquid-based cytology sample. [ABSTRACT FROM AUTHOR]

The development of new strategies for the analysis of nucleic acids has gained momentum due to the increased interest in using these biomolecules as drugs or drug targets. The application of new mass spectrometry ion activation techniques and the optimization of separation methods including liquid chromatography, capillary electrophoresis, and ion mobility have allowed more detailed characterization of nucleic acids and oligonucleotide therapeutics including confirmation of sequence, localization of modifications and interaction sites, and structural analysis as well as identification of failed sequences and degradation products. This review will cover tandem mass spectrometry methods as well as the recent developments in liquid chromatography, capillary electrophoresis, and ion mobility coupled to mass spectrometry for the analysis of nucleic acids and oligonucleotides. [ABSTRACT FROM AUTHOR]

The natural four-letter genetic alphabet, comprised of just two base pairs (dA-dT and dG-dC), is conserved throughout all life, and its expansion by the development of a third, unnatural base pair has emerged as a central goal of chemical and synthetic biology. We recently developed a class of candidate unnatural base pairs, exemplified by the pair formed between d5SICS and dNaM. Here, we examine the PCR amplification of DNA containing one or more d5SICS-dNaM pairs in a wide variety of sequence contexts. Under standard conditions, we show that this DNA may be amplified with high efficiency and greater than 99.9% fidelity. To more rigorously explore potential sequence effects, we used deep sequencing to characterize a library of templates containing the unnatural base pair as a function of amplification. We found that the unnatural base pair is efficiently replicated with high fidelity in virtually all sequence contexts. The results show that, for PCR and PCR-based applications, d5SICS-dNaM is functionally equivalent to a natural base pair, and when combined with dA-dT and dG-dC, it provides a fully functional six-letter genetic alphabet. [ABSTRACT FROM AUTHOR]

Both antisense oligonucleotides (ASODN) and small interfering RNA (siRNA) have enormous potential to selectively silence specific cancer-related genes and could therefore be developed to be important therapeutic anti-cancer drugs. The use of nanotechnology may allow for significant advancement of the therapeutic potential of ASODN and siRNA, due to improved pharmacokinetics, bio-distribution and tissue specific targeted therapy. In this mini-review, we have discussed the advantages of using a nanocarrier such as a multimodal quantum rod (QR) complexed with siRNA for gene delivery. Comparisons are made between ASODN and siRNA therapeutic efficacies in the context of cancer and the enormous application potential of nanotechnology in oncotherapy is discussed. We have shown that a QR-interleukin-8 (IL-8) siRNA nanoplex can effectively silence IL-8 gene expression in the PC-3 prostate cancer cells with no significant toxicity. Thus, nanocarriers such as QRs can help translate the potent effects of ASODN/siRNA into a clinically viable anti-cancer therapy. Drug delivery for cancer therapy, with the aid of nanotechnology is one of the major translational aspects of nanomedicine, and efficient delivery of chemotherapy drugs and gene therapy drugs or their co-delivery continue to be a major focus of nanomedicine research. [ABSTRACT FROM AUTHOR]

Gram-negative bacteria have the ability to produce outer membrane-derived vesicles (OMVs) that are released into the extracellular milieu. Even though this intriguing phenomenon is well-known since many years, various aspects of bacterial OMVs are not fully described and are still in the process of being characterized in detail. One major reason for this is that depending on the bacterial species and its respective ecological niche, OMVs exhibit an enormous functional diversity. Research of the past years has clearly shown that OMVs of many pathogenic bacteria contribute to the virulence potential by enriching virulence factors and delivering them over long distances, superseding direct bacterial contact with their host. The subsequent interaction of OMVs with the host can occur at different levels regarding the type of immune response or the target cell type and may lead to different outcomes ranging from non-immunogenic activation or a pro-inflammatory response to cytotoxicity. In contrast to being virulence factors, OMVs are used for vaccination purposes in the combat against bacterial pathogens, and recent research thus is focused on to indirectly aim these versatile bacterial weapons against themselves. [ABSTRACT FROM AUTHOR]

The article presents a study reviewing all data available in the literature on the detection of spirochetes in Alzheimer's disease (AD) in order to analyze the association and causal relationship between spirochetes and AD following an established criteria. It has been shown by the study that there is a statistically significant association between spirochetes and AD. Also, borrelia burgdorferi was detected in the brain in 25.3% of AD cases which were analyzed in the study.