Using GPCRs as Molecular Beacons to Target Ovarian Cancer with Nanomedicines.

The five-year survival rate for women with ovarian cancer is very poor despite radical cytoreductive surgery and chemotherapy. Although most patients initially respond to platinum-based chemotherapy, the majority experience recurrence and ultimately develop chemoresistance, resulting in fatal outcomes. The current administration of cytotoxic compounds is hampered by dose-limiting severe adverse effects. There is an unmet clinical need for targeted drug delivery systems that transport chemotherapeutics selectively to tumor cells while minimizing off-target toxicity. G protein-coupled receptors (GPCRs) are the largest family of membrane receptors, and many are overexpressed in solid tumors, including ovarian cancer. This review summarizes the progress in engineered nanoparticle research for drug delivery for ovarian cancer and discusses the potential use of GPCRs as molecular entry points to deliver anti-cancer compounds into ovarian cancer cells. A newly emerging treatment paradigm could be the personalized design of nanomedicines on a case-by-case basis.

Development of a Unique Rapid Test to Detect Anti-bodies Directed Against an Extended RBD of SARS-CoV-2 Spike Protein.

Serological testing for antibodies directed against SARS-CoV-2 in patients may serve as a diagnostic tool to verify a previous infection and as surrogate for an elicited humoral immune response, ideally conferring immunity after infection or vaccination. Here, we present the recombinant expression of an extended receptor binding domain (RBD) of the SARS-CoV-2 Spike protein used as capture antigen in a unique rapid immunoassay to detect the presence of RBD binding antibodies with high sensitivity and specificity. As currently available vaccines focus on the Spike RBD as target, the developed test can also be used to monitor a successful immune response after vaccination with an RBD based vaccine.

Systematic Meta-Analysis Identifies Co-Expressed Kinases and GPCRs in Ovarian Cancer Tissues Revealing a Potential for Targeted Kinase Inhibitor Delivery.

The use of many anticancer drugs is problematic due to severe adverse effects. While the recent clinical launch of several kinase inhibitors led to tremendous progress, these targeted agents tend to be of non-specific nature within the kinase target class. Moreover, target mediated adverse effects limit the exploitation of some very promising kinase targets, including mitotic kinases. A future strategy will be the development of nanocarrier-based systems for the active delivery of kinase inhibitors using cancer specific surface receptors. The G-protein-coupled-receptors (GPCRs) represent the largest cell surface receptor family and some members are known to be frequently overexpressed in various cancer types. In the presented study, we used ovarian cancer tissues as an example to systematically identify concurrently overexpressed GPCRs and kinases. The rationale of this approach will guide the future design of nanoparticles, which will dock to GPCRs on cancer cells via specific ligands and deliver anticancer compounds after receptor mediated internalization. In addition to this, the approach is expected to be most effective by matching the inhibitor profiles of the delivered kinase inhibitors to the observed kinase gene expression profiles. We validated the suggested strategy in a meta-analysis, revealing overexpression of selected GPCRs and kinases in individual samples of a large ovarian cancer data set. The presented data demonstrate a large untapped potential for personalized cancer therapy using high-end targeted nanopharmaceuticals with kinase inhibitors.

Development of a DNA-based Assay to Detect and Quantify Tropane Alkaloids Producing Thornapple Contaminations in Processed Food.

Contaminates such as pesticides, toxic molecules of natural origin, genetically modified organisms and others can occur in processed food, especially if the main ingredient grows in open fields exposed to the environment. In particular, some health threatening toxic compounds are natural ingredients of plants that grow wild next to vegetables intended for consumption and can therefore enter the crop yield and stay there undetected. The tropane alkaloids-containing nightshade thornapple Datura stramonium, often grows in close vicinity to millet (Panicum miliaceum) a widely cultivated cereal, representing an important nutrient source in different countries of Asia and Africa. Discriminating thornapple from millet during harvest is not easy and consequently, millet-containing food products are often contaminated with tropane alkaloids from thornapple. In this work, two DNA specific hydrolysis probe qPCR methods were developed for Datura stramonium and Panicum miliaceum in order to detect thornapple contamination in millet-containing food products. The specificity and sensitivity of the developed assay system allows for its application in screenings during food product testing.

Large set data mining reveals overexpressed GPCRs in prostate and breast cancer: potential for active targeting with engineered anti-cancer nanomedicines.

Over 800 G-protein-coupled receptors (GPCRs) are encoded by the human genome and many are overexpressed in tumors. GPCRs are triggered by ligand molecules outside the cell and activate internal signal transduction pathways driving cellular responses. The receptor signals are desensitized by receptor internalization and this mechanism can be exploited for the specific delivery of ligand-linked drug molecules directly into cells. Detailed expression analysis in cancer tissue can inform the design of GPCR-ligand decorated drug carriers for active tumor cell targeting. The active targeting process utilizes ligand receptor interactions leading to binding and in most cases internalization of the ligand-attached drug carrier resulting in effective targeting of cancer cells. In this report public microarray data from the Gene Expression Omnibus (GEO) repository was used to identify overexpressed GPCRs in prostate and breast cancer tissues. The analyzed data confirmed previously known cancer receptor associations and identified novel candidates for potential active targeting. Prioritization of the identified targeting receptors is also presented based on high expression levels and frequencies in cancer samples but low expression in healthy tissue. Finally, some selected examples were used in ligand docking studies to assess the feasibility for chemical conjugation to drug nanocarriers without interference of receptor binding and activation. The presented data demonstrate a large untapped potential to improve efficacy and safety of current and future anti-cancer compounds through active targeting of GPCRs on cancer cells.

Yeast-based assays for screening 11ß-HSD1 inhibitors.

BACKGROUND: Intracellular metabolism of glucocorticoid hormones plays an important role in the pathogenesis of metabolic syndrome and regulates, among many physiological processes, collagen metabolism in skin. At the peripheral level the concentration of active glucocorticoids is mainly regulated by the 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) enzyme, involved in the conversion of cortisone into the biologically active hormone cortisol. Cortisol interacts with the glucocorticoid receptor and regulates the expression of different classes of genes within the nucleus. Due to its implication in glucocorticoid metabolism, the inhibition of 11ß-HSD1 activity has become a dominant strategy for the treatment of metabolic syndrome. Moreover, inhibitors of this target enzyme can be used for development of formulations to counteract skin ageing. Here we present the construction of two yeast cell based assays that can be used for the screening of novel 11ß-HSD1 inhibitors. RESULTS: The yeast Saccharomyces cerevisiae is used as a host organism for the expression of human 11ß-HSD1 as well as a genetically encoded assay system that allows intracellular screening of molecules with 11ß-HSD1 inhibitory activity. As proof of concept the correlation between 11ß-HSD1 inhibition and fluorescent output signals was successfully tested with increasing concentrations of carbenoxolone and tanshinone IIA, two known 11ß-HSD1 inhibitors. The first assay detects a decrease in fluorescence upon 11ß-HSD1 inhibition, whereas the second assay relies on stabilization of yEGFP upon inhibition of 11ß-HSD1, resulting in a positive read-out and thus minimizing the rate of false positives sometimes associated with read-outs based on loss of signals. Specific inhibition of the ABC transporter Pdr5p improves the sensitivity of the assay strains to cortisone concentrations by up to 60 times. CONCLUSIONS: Our yeast assay strains provide a cost-efficient and easy to handle alternative to other currently available assays for the screening of 11ß-HSD1 inhibitors. These assays are designed for an initial fast screening of large numbers of compounds and enable the selection of cell permeable molecules with target inhibitory activity, before proceeding to more advanced selection processes. Moreover, they can be employed in yeast synthetic biology platforms to reconstitute heterologous biosynthetic pathways of drug-relevant scaffolds for simultaneous synthesis and screening of 11ß-HSD1 inhibitors at intracellular level.

Unconventional P-35S sequence identified in genetically modified maize.

The Cauliflower Mosaic Virus 35S promoter sequence, CaMV P-35S, is one of several commonly used genetic targets to detect genetically modified maize and is found in most GMOs. In this research we report the finding of an alternative P-35S sequence and its incidence in GM maize marketed in Jordan. The primer pair normally used to amplify a 123 bp DNA fragment of the CaMV P-35S promoter in GMOs also amplified a previously undetected alternative sequence of CaMV P-35S in GM maize samples which we term V3. The amplified V3 sequence comprises 386 base pairs and was not found in the standard wild-type maize, MON810 and MON 863 GM maize. The identified GM maize samples carrying the V3 sequence were found free of CaMV when compared with CaMV infected brown mustard sample. The data of sequence alignment analysis of the V3 genetic element showed 90% similarity with the matching P-35S sequence of the cauliflower mosaic virus isolate CabbB-JI and 99% similarity with matching P-35S sequences found in several binary plant vectors, of which the binary vector locus JQ693018 is one example. The current study showed an increase of 44% in the incidence of the identified 386 bp sequence in GM maize sold in Jordan's markets during the period 2009 and 2012.

[Fixation of cells for analysis by laser microdissection--comparative studies in forensic trace material]. / Fixierung von Zellen zur Analyse mittels Laser-Mikrodissektion: Vergleichende Untersuchungen an forensischem Spurenmaterial.

This paper is focused on the preparation of samples for laser microdissection (LM) in forensic casework. In forensic genetics, it is essential to preserve and separate cellular traces during sample preparation, as they are usually gathered in very small amounts and are often contaminated with undesired cells. This is made possible by laser microdissection, a technique developed to cut cells or tissue of a certain type from a microscopical specimen by UV laser and catapult them directly into a PCR reactor. This method minimizes the risk of getting inconclusive, mixed DNA profiles due to contamination by foreign DNA and also supplies information about the cellular origin of a DNA profile. A method for optimized fixation and staining of spermatozoa for laser microdissection was established. Four different fixation methods combined with two staining methods were tested on two different microscope slides. Moreover, the effect of a blocker pen to contain the specimen on the slide was investigated.

[Recovery of blood traces with scenesafe FAST tape]. / Sicherung von Blutspuren mit Scenesafe FAST Tape.

For complete and conclusive DNA profiling a sufficient amount of DNA must be available. For that, biological traces are recovered from crime scenes using special trace recovery material. The current method to collect biological traces for DNA analysis is to wipe them off with cotton swabs. However, for a few years the use of adhesive tapes has also been described for the recovery of evidence. In 2009, an adhesive tape was launched which was specially developed for the collection of biological traces. This product called Scenesafe FAST Tape (SSF) was investigated in this work to give recommendations for its use in evidence recovery. The results of this work show that the DNA can be extracted from the SSF very well. However, the tapes seem less suitable for direct use at the crime scene, as they are not flexible enough for adaptation to different crime scene conditions and the risk of contamination is higher than when collecting evidence with cotton swabs. Neither SSF nor cotton swabs are optimal for all requirements. The best method to recover biological evidence is still dependent on the surface material and the circumstances at the crime scene.

Diagnosis of prion diseases.

Prion diseases are usually diagnosed clinically and confirmed by post-mortem histopathological examination of brain tissue. The only reliable molecular marker for prion diseases is PrP(Sc), the pathological conformer of the prion protein that accumulates in the central nervous system and, to a lesser extent, in lymphoreticular tissues. For BSE, several commercial diagnostic kits based on the post-mortem immunochemical detection of PrP(Sc) in brain tissue are now available. These rapid screening tests have been used in active surveillance of BSE and have greatly improved the detection of infected cattle before their entry into the human food chain. At present, no diagnostic test exists for the detection of prion diseases in live animals or humans. New diagnostic techniques aimed at increasing sensitivity and specificity of PrP(Sc) detection in body fluids and at identifying novel surrogate markers are under development. In this report, we review the classical diagnostic methods as well as present and future tools for the diagnosis of prion diseases.

Validation of a luminescence immunoassay for the detection of PrP(Sc) in brain homogenate.

A luminescence immunoassay (LIA) was developed for the diagnosis of bovine spongiform encephalopathy (BSE) in brain tissue using two different monoclonal antibodies for capture and detection of the protease-resistant fragment of the pathological prion protein (PrP27-30). PrP27-30 currently represents the most reliable marker for the infectious particle (denominated prion) causing transmissible spongiform encephalopathies (TSEs). Internal and official validation studies of this assay are described using brain homogenates from ascertained BSE positive and negative cows. Using more than 300 positive and 1400 negative bovine or ovine samples, an excellent sensitivity and specificity of 100% were demonstrated. More than 1000-fold dilutions of a BSE positive homogenate still resulted in a clear positive signal. In combination with a simple homogenisation procedure for the preparation of the samples, this assay lends itself for large scale screening of cattle and sheep for TSEs using complete automation of the process.