Assembly and use of high-density recombinant peptide chips for large-scale ligand screening is a practical alternative to synthetic peptide libraries.

BACKGROUND: Recombinant peptide chips could constitute a versatile complementation to state-of-the-art in situ (chemical on-chip) synthesis, particle-based printing, or pre-manufactured peptide spotting. Bottlenecks still impeding a routine implementation - from restricted peptide lengths, low diversity and low array densities to high costs - could so be overcome. METHODS: To assess overall performance, we assembled recombinant chips composed of 38,400 individual peptide spots on the area of a standard 96-well microtiter plate from comprehensive, highly diverse (>107 single clones) short random peptide libraries. RESULTS: Screening of altogether 476,160 clones against Streptavidin uncovered 2 discrete new binders: a characteristic HPQ-motif containing VSHPQAPF and a cyclic CSGSYGSC peptide. Interactions were technically confirmed by fluorescence polarization as well as biolayer-interferometry, and their potential suitability as novel detection tags evaluated by detection of a peptide-fused exemplary test protein. CONCLUSION: From our data we conclude that the presented technical pipeline can reliably identify novel hits, useful as first-generation binders or templates for subsequent ligand design plus engineering.

Structural analysis and interaction studies of acyl-carrier protein (acpP) of Staphylococcus aureus, an extraordinarily thermally stable protein.

Acyl-carrier-protein (acpP) is an essential protein in fatty acid biosynthesis of Staphylococcus aureus [Cronan, J.E. and Thomas, J. (2009). Complex enzymes in microbial natural product biosynthesis, part B: polyketides, aminocoumarins and carbohydrates. METHOD: Enzymol. 459, 395-433; Halavaty, A.S., Kim, Y., Minasov, G., Shuvalova, L., Dubrovska, I., Winsor, J., Zhou, M., Onopriyenko, O., Skarina, T., Papazisi, L., et al. (2012). Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria. Acta Crystallogr. Sect. D Biol. Crystallogr. 68, 1359-1370]. The inactive apo-form is converted to the active holo-enzyme by acyl-carrier protein synthase (acpS) through addition of a 4'-phosphopantetheine group from coenzyme A to a conserved serine residue of acpP [Flugel, R.S., Hwangbo, Y., Lambalot, R.H., Cronan, J.E., and Walsh, C.T. (2000). Holo-(acyl-carrier protein) synthase and phosphopantetheinyl transfer in Escherichia coli. J. Biol. Chem. 275, 959-968; Lambalot, R.H. and Walsh, C.T. (1995). Cloning, overproduction, and characterization of the Escherichia coli holo-acyl-carrier protein synthase. J. Biol. Chem. 270, 24658-24661]. Once activated, acpP acts as an anchor for the growing fatty acid chain. Structural data from X-ray crystallographic analysis reveals that, despite its small size (8 kDa), acpP adopts a distinct, mostly α-helical structure when complexed with acpS [Halavaty, A.S., Kim, Y., Minasov, G., Shuvalova, L., Dubrovska, I., Winsor, J., Zhou, M., Onopriyenko, O., Skarina, T., Papazisi, L., et al. (2012). Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria. Acta Crystallogr. Sect. D Biol. Crystallogr. 68, 1359-1370; Byers, D.M. and Gong, H. (2007). Acyl carrier protein: structure-function relationships in a conserved multifunctional protein family. Biochem. Cell Biol. 85, 649-662]. We expressed and purified recombinant, active S. aureus acpP from Escherichia coli and mimicked the beginning of fatty acid biosynthesis by employing an [14C]-acp loading assay. Surprisingly, acpP remained functional even after heat treatment at 95°C for up to 10 min. NMR data from 2D-HSQC experiments as well as interaction studies with acpS confirmed that acpP is structured and active both before and after heat treatment, with no significant differences between the two. Thus, our data suggest that S. aureus acpP is a highly stable protein capable of maintaining its structure at high temperatures.

Design, Synthesis, and Cytotoxicity of 5-Fluoro-2-methyl-6-(4-aryl-piperazin-1-yl) Benzoxazoles.

To design new compounds suitable as starting points for anticancer drug development, we have synthesized a novel series of benzoxazoles with pharmaceutically advantageous piperazine and fluorine moieties attached to them. The newly synthesized benzoxazoles and their corresponding precursors were evaluated for cytotoxicity on human A-549 lung carcinoma cells and non-cancer HepaRG hepatocyes. Some of these new benzoxazoles show potential anticancer activity, while two of the intermediates show lung cancer selective properties at low concentrations where healthy cells are unaffected, indicating a selectivity window for anticancer compounds.

Application of DNA chip scanning technology for automatic detection of Chlamydia trachomatis and Chlamydia pneumoniae inclusions.

Chlamydiae are obligate intracellular bacteria that propagate in the inclusion, a specific niche inside the host cell. The standard method for counting chlamydiae is immunofluorescent staining and manual counting of chlamydial inclusions. High- or medium-throughput estimation of the reduction in chlamydial inclusions should be the basis of testing antichlamydial compounds and other drugs that positively or negatively influence chlamydial growth, yet low-throughput manual counting is the common approach. To overcome the time-consuming and subjective manual counting, we developed an automatic inclusion-counting system based on a commercially available DNA chip scanner. Fluorescently labeled inclusions are detected by the scanner, and the image is processed by ChlamyCount, a custom plug-in of the ImageJ software environment. ChlamyCount was able to measure the inclusion counts over a 1-log-unit dynamic range with a high correlation to the theoretical counts. ChlamyCount was capable of accurately determining the MICs of the novel antimicrobial compound PCC00213 and the already known antichlamydial antibiotics moxifloxacin and tetracycline. ChlamyCount was also able to measure the chlamydial growth-altering effect of drugs that influence host-bacterium interaction, such as gamma interferon, DEAE-dextran, and cycloheximide. ChlamyCount is an easily adaptable system for testing antichlamydial antimicrobials and other compounds that influence Chlamydia-host interactions.

A de novo-designed antimicrobial peptide with activity against multiresistant Staphylococcus aureus acting on RsbW kinase.

Antimicrobial peptides are a promising complement to common antibiotics, development of resistance to which is a growing problem. Here we present a de novo-designed peptide, SP1-1 (RKKRLKLLKRLL-NH2), with antimicrobial activity against multiresistant Staphylococcus aureus (minimal inhibitory concentration: 6.25 µM). Elucidation of the mode of action of this peptide revealed a strong interaction with RsbW kinase (Kd: 6.01±2.73 nM), a serine kinase negatively regulating the activity of the transcription factor σB (SigB). SP1-1 binding and functional modulation of RsbW were shown in vitro by a combination of biochemical, molecular, and biophysical methods, which were further genetically evidenced in vivo by analysis of S. aureus ΔsigB deletion mutants. Intracellular localization of the peptide was demonstrated using nanometer-scaled secondary ion mass spectrometry. Moreover, microarray analysis revealed that transcription of numerous genes, involved in cell wall and amino acid metabolism, transport mechanisms, virulence, and pigmentation, is affected. Interestingly, several WalR binding motif containing genes are induced by SP1-1. In sum, the designed peptide SP1-1 seems to have multiple modes of action, including inhibition of a kinase, and therefore might contribute to the development of new antibacterial compounds, giving bacterial kinase inhibition a closer inspection.

Imbalance of intermediate filament component keratin 14 contributes to increased stress signalling in epidermolysis bullosa simplex.

An important characteristic of epidermolysis bullosa simplex Dowling-Meara (EBS-DM) keratinocytes is the increased level of Jun N-terminal kinase (JNK) stress signalling, which is thought to contribute to the disease phenotype. In this work, we report on the dramatic up-regulation of cytokeratin 14 (K14) in the EBS-DM model cell line KEB7 at both the transcriptional and translational levels, which is noteworthy because KEB7 patient cells are heterozygous for a missense mutation (R125P) in K14. By performing functional assays, we show a direct link between overexpressed wild-type K14 and increased JNK signalling in healthy, immortalized keratinocytes. This observation led us to hypothesize a positive feedback model in which mutant (R125P) K14 triggers JNK signalling, leading to increased AP1-dependent expression of K14, which in turn amplifies JNK signalling further. We therefore suggest that an imbalance of cytoplasmic K14 monomers and K14 incorporated into the intermediate filament (IF) network leads to elevated stress signalling, potentially altering IF dynamics by phosphorylation, which as a side effect, weakens EBS-DM keratinocytes.

Quantitative real-time PCR as a sensitive protein-protein interaction quantification method and a partial solution for non-accessible autoactivator and false-negative molecule analysis in the yeast two-hybrid system.

Many functional proteomic experiments make use of high-throughput technologies such as mass spectrometry combined with two-dimensional polyacrylamide gel electrophoresis and the yeast two-hybrid (Y2H) system. Currently there are even automated versions of the Y2H system available that can be used for proteome-wide research. The Y2H system has the capacity to deliver a profusion of Y2H positive colonies from a single library screen. However, subsequent analysis of these numerous primary candidates with complementary methods can be overwhelming. Therefore, a method to select the most promising candidates with strong interaction properties might be useful to reduce the number of candidates requiring further analysis. The method described here offers a new way of quantifying and rating the performance of positive Y2H candidates. The novelty lies in the detection and measurement of mRNA expression instead of proteins or conventional Y2H genetic reporters. This method correlates well with the direct genetic reporter readouts usually used in the Y2H system, and has greater sensitivity for detecting and quantifying protein-protein interactions (PPIs) than the conventional Y2H system, as demonstrated by detection of the Y2H false-negative PPI of RXR/PPARG. Approximately 20% of all proteins are not suitable for the Y2H system, the so-called autoactivators. A further advantage of this method is the possibility to evaluate molecules that usually cannot be analyzed in the Y2H system, exemplified by a VDR-LXXLL motif peptide interaction.

Construction of a highly flexible and comprehensive gene collection representing the ORFeome of the human pathogen Chlamydia pneumoniae.

BACKGROUND: The Gram-negative bacterium Chlamydia pneumoniae (Cpn) is the leading intracellular human pathogen responsible for respiratory infections such as pneumonia and bronchitis. Basic and applied research in pathogen biology, especially the elaboration of new mechanism-based anti-pathogen strategies, target discovery and drug development, rely heavily on the availability of the entire set of pathogen open reading frames, the ORFeome. The ORFeome of Cpn will enable genome- and proteome-wide systematic analysis of Cpn, which will improve our understanding of the molecular networks and mechanisms underlying and governing its pathogenesis. RESULTS: Here we report the construction of a comprehensive gene collection covering 98.5% of the 1052 predicted and verified ORFs of Cpn (Chlamydia pneumoniae strain CWL029) in Gateway(®) 'entry' vectors. Based on genomic DNA isolated from the vascular chlamydial strain CV-6, we constructed an ORFeome library that contains 869 unique Gateway(®) entry clones (83% coverage) and an additional 168 PCR-verified 'pooled' entry clones, reaching an overall coverage of ~98.5% of the predicted CWL029 ORFs. The high quality of the ORFeome library was verified by PCR-gel electrophoresis and DNA sequencing, and its functionality was demonstrated by expressing panels of recombinant proteins in Escherichia coli and by genome-wide protein interaction analysis for a test set of three Cpn virulence factors in a yeast 2-hybrid system. The ORFeome is available in different configurations of resource stocks, PCR-products, purified plasmid DNA, and living cultures of E. coli harboring the desired entry clone or pooled entry clones. All resources are available in 96-well microtiterplates. CONCLUSION: This first ORFeome library for Cpn provides an essential new tool for this important pathogen. The high coverage of entry clones will enable a systems biology approach for Cpn or host-pathogen analysis. The high yield of recombinant proteins and the promising interactors for Cpn virulence factors described here demonstrate the possibilities for proteome-wide studies.

PIM-1 kinase interacts with the DNA binding domain of the vitamin D receptor: a further kinase implicated in 1,25-(OH)2D3 signaling.

BACKGROUND: The vitamin D3 receptor (VDR) is responsible for mediating the pleiotropic and, in part, cell-type-specific effects of 1,25-dihydroxyvitamin D3 (calcitriol) on the cardiovascular and the muscle system, on the bone development and maintenance, mineral homeostasis, cell proliferation, cell differentiation, vitamin D metabolism, and immune response modulation. RESULTS: Based on data obtained from genome-wide yeast two-hybrid screenings, domain mapping studies, intracellular co-localization approaches as well as reporter transcription assay measurements, we show here that the C-terminus of human PIM-1 kinase isoform2 (amino acid residues 135-313), a serine/threonine kinase of the calcium/calmodulin-regulated kinase family, directly interacts with VDR through the receptor's DNA-binding domain. We further demonstrate that PIM-1 modulates calcitriol signaling in HaCaT keratinocytes by enhancing both endogenous calcitriol response gene transcription (osteopontin) and an extrachromosomal DR3 reporter response. CONCLUSION: These results, taken together with previous reports of involvement of kinase pathways in VDR transactivation, underscore the biological relevance of this novel protein-protein interaction.

Gene expression analysis of an epidermolysis bullosa simplex Dowling-Meara cell line by subtractive hybridization: recapitulation of cellular differentiation, migration and wound healing.

An intact keratin 5/keratin 14 intermediate filament cytoskeleton is vital for the integrity of basal keratinocytes and for the development and maintenance of epidermal structures. In patients with epidermolysis bullosa simplex Dowling-Meara (EBS-DM), heterozygous mutations in the keratin 14 gene in keratinocytes cause a cytoskeletal collapse leading to fragile cells susceptible to cellular stress. The primary aim of this work was to extend analysis of differentially expressed genes in an EBS-DM model cell line to obtain insights into the molecular consequences resulting from the keratin 14 mutation. In a first step, suppression subtractive hybridization (SSH), a powerful technology to enrich for differentially expressed genes, was used to identify genes whose up-regulation may be a direct or indirect result of the keratin 14 mutation, R125P. We discovered 55 candidate genes (SSH genes) that were further analysed by RTq-PCR. Of the 55 SSH genes, 14 (25.45%) were found to be congruently up-regulated. Bioinformatic analysis revealed significant enrichment of genes regulating epidermal development, migration, apoptosis and wound healing.

Proteomic profiling reveals a catalogue of new candidate proteins for human skin aging.

Studies of skin aging are usually performed at the genomic level by investigating differentially regulated genes identified through subtractive hybridization or microarray analyses. In contrast, relatively few studies have investigated changes in protein expression of aged skin using proteomic profiling by two-dimensional (2-D) gel electrophoresis and mass spectrometry, although this approach at the protein level is suggested to reflect more accurately the aging phenotype. We undertook such a proteomic analysis of intrinsic human skin aging by quantifying proteins extracted and fluorescently labeled from sun-protected human foreskin samples pooled from 'young' and 'old' men. In addition, we analyzed these candidate gene products by 1-D and 2-D western blotting to obtain corroborative protein expression data, and by both real-time PCR (RT-PCR) and microarray analyses to confirm expression at the mRNA level. We discovered 30 putative proteins for skin aging, including previously unrecognized, post-translationally regulated candidates such as phosphatidyl-ethanolamine binding protein (PEBP) and carbonic anhydrase 1 (CA1).

K16 is a further new candidate for homotypic intermediate filament protein interactions.

Keratin filaments form obligatory heterodimers consisting of one type I and one type II keratin that build the intermediate filaments (IF). These filaments mediate resilience and mechanical strength to epithelial cells and maintain tissue integrity. Specific type I/type II pairs are co-expressed in vivo and serve as markers for distinct tissue layers and cell differentiation states. Heterodimerization has been regarded the undisrupted hallmark of IF. We show now that recombinantly expressed cytokeratin 16 (K16) interacts with itself and forms homodimers even in denaturating SDS-PAGE analysis. Detailed FRET experiments in HaCaT keratinocytes were in accordance with our in vitro observations and showed clearly that K16 is able to form strong homodimers. Homotypic keratin interactions has been previously shown for keratin 17 (K17) and keratin 18 (K18) by Schnabel et al. (Biochim Biophys Acta, 1998: 1403: 158), and we now proved K16 to be the third type I keratin that is able to form homodimers.

Management and Prevention Strategies for Non-communicable Diseases (NCDs) and Their Risk Factors.

Non-communicable diseases (NCDs) are of increasing concern for society and national governments, as well as globally due to their high mortality rate. The main risk factors of NCDs can be classified into the categories of self-management, genetic factors, environmental factors, factors of medical conditions, and socio-demographic factors. The main focus is on the elements of self-management and to reach a consensus about the influence of food on risk management and actions toward the prevention of NCDs at all stages of life. Nutrition interventions are essential in managing the risk of NCDs. As they are of the utmost importance, this review highlights NCDs and their risk factors and outlines several common prevention strategies. We foresee that the best prevention management strategy will include individual (lifestyle management), societal (awareness management), national (health policy decisions), and global (health strategy) elements, with target actions, such as multi-sectoral partnership, knowledge and information management, and innovations. The most effective preventative strategy is the one that leads to changes in lifestyle with respect to diet, physical activities, cessation of smoking, and the control of metabolic disorders.

Improved homology-driven computational validation of protein-protein interactions motivated by the evolutionary gene duplication and divergence hypothesis.

BACKGROUND: Protein-protein interaction (PPI) data sets generated by high-throughput experiments are contaminated by large numbers of erroneous PPIs. Therefore, computational methods for PPI validation are necessary to improve the quality of such data sets. Against the background of the theory that most extant PPIs arose as a consequence of gene duplication, the sensitive search for homologous PPIs, i.e. for PPIs descending from a common ancestral PPI, should be a successful strategy for PPI validation. RESULTS: To validate an experimentally observed PPI, we combine FASTA and PSI-BLAST to perform a sensitive sequence-based search for pairs of interacting homologous proteins within a large, integrated PPI database. A novel scoring scheme that incorporates both quality and quantity of all observed matches allows us (1) to consider also tentative paralogs and orthologs in this analysis and (2) to combine search results from more than one homology detection method. ROC curves illustrate the high efficacy of this approach and its improvement over other homology-based validation methods. CONCLUSION: New PPIs are primarily derived from preexisting PPIs and not invented de novo. Thus, the hallmark of true PPIs is the existence of homologous PPIs. The sensitive search for homologous PPIs within a large body of known PPIs is an efficient strategy to separate biologically relevant PPIs from the many spurious PPIs reported by high-throughput experiments.

Direct qPCR is a sensitive approach to detect Mycoplasma contamination in U937 cell cultures.

OBJECTIVE: We aim to directly detect Mycoplasma DNA in a U937 suspension cell culture without using DNA purification. In order to make Mycoplasma contamination monitoring easier, we optimized a commercially available quantitative PCR (qPCR)-based detection kit. We compared the sensitivity of direct qPCR against qPCR with a purified DNA template. RESULTS: Our findings indicate that qPCR worked optimally with a 6 µl sample volume and a 52 °C annealing-extension temperature. We were able to decrease the annealing-extension step time from 60 to 20 s without any major decrease in reaction sensitivity. The total cycle time of optimized direct qPCR was 65 min. The optimized qPCR protocol was used to detect Mycoplasma DNA before and after DNA purification. Our findings indicate that direct qPCR had a higher sensitivity than regular qPCR. Ct levels produced by direct qPCR with 6 µl templates were almost identical to Ct levels produced by regular qPCR with DNA purified from a 60 µl cell culture sample (23.42 vs 23.49 average Ct levels, respectively). The optimized direct qPCR protocol was successfully applied to monitor the elimination of Mycoplasma contamination from U937 cell cultures.

A Sight to Wheat Bran: High Value-Added Products.

Recently more consideration has been given to the use of renewable materials and agricultural residues. Wheat production is increasing yearly and correspondingly, the volume of by-products from the wheat process is increasing, as well. It is important to find the use of the residuals for higher value-added products, and not just for the food industry or animal feed purposes as it is happening now. Agricultural residue of the roller milled wheat grain is a wheat bran description. The low-cost of wheat bran and its composition assortment provides a good source of substrate for various enzymes and organic acids production and other biotechnological applications. The main purpose of this review article is to look into recent trends, developments, and applications of wheat bran.

The ORFeome of Staphylococcus aureus v 1.1.

BACKGROUND: The bacterium Staphylococcus aureus causes significant morbidity and mortality in humans, primarily due to the emergence of strains that are resistant to antibiotics - notably methicillin-resistant S. aureus (MRSA) isolates. Development of effective strategies for the control and treatment of MRSA infections may best be achieved through 'omics' approaches, which first requires cloning the entire set of S. aureus' protein-encoding open reading frames (ORFs), or ORFeome. RESULTS: The complete genome sequence of S. aureus strain Mu50 has 2697 predicted protein-coding ORFs. Based on the sequence of this strain we designed PCR primers to construct from an S. aureus (non-MRSA) clinical isolate an ORFeome library that contains 2562 unique Gateway entry clones (95% coverage), each corresponding to a defined ORF. The high quality of the ORFeome library was verified by DNA sequencing and PCR amplification, and its functionality was demonstrated by expressing recombinant proteins and observing protein interactions in a yeast 2-hybrid homodimerization screen. CONCLUSION: This first ORFeome library for S. aureus provides an essential new tool for investigating the systems biology of this important pathogen.

Construction of a reading frame-independent yeast two-hybrid vector system for site-specific recombinational cloning and protein interaction screening.

The yeast two-hybrid (Y2H) system is a powerful method to identify protein-protein inter-actions (PPI) in vivo, requiring minimal prior information of the putative interactors. The time and effort required for each experiment can be significantly reduced if the "bait" and the "prey" proteins are cloned into specific recombination-amenable two-hybrid vectors. We describe the construction of a reading frame-independent vector system for Y2H PPI studies. The described vector system knits together the advantages of site-specific recombination cloning with the Y2H system. The produced plasmids enable recombination-based cloning of genes or gene fragments in all possible reading frames into Y2H library vectors. Thus, Y2H screening libraries can be rapidly constructed and will present more amino termini in the correct reading frame. Additionally, advantageous for small-scale Y2H studies, there is no need to know the natural reading frame of the genes of interest, because the bait and prey genes can be transferred into the vectors by a single reaction and are present in all possible reading frames. Since the Y2H system per se is a positive selection system, only pairs of bait and prey genes harboring the correct reading frames will emerge. We tested the new vectors within the Y2H system and demonstrated full functionality without any undesired effects on the Y2H system itself. Besides the vector construction, we investigated the utility of the system for Y2H analysis and demonstrated clearly its practicability in genome-wide Y2H screenings and the advantage of using additional reading-frame Y2H cDNA libraries. We performed a series of genome-wide Y2H library screenings with the human vitamin D receptor protein (VDR) as bait. We investigated: (i) whether more protein interactors are found by using three instead of one reading-frame destination vectors; (ii) how much overlap between the different reading-frame libraries exists; and (iii) the rate of possible additional autoactivators. We conclude that our vectors deliver significantly more interactors and outperform a single reading-frame library. This new system could enable simple and fast large-scale PPI studies and the construction of high-quality screening libraries.

A direct quantitative PCR-based measurement of herpes simplex virus susceptibility to antiviral drugs and neutralizing antibodies.

Herpes simplex viruses (HSV) are common human pathogens that can cause painful but benign manifestations and recurrent complaints, but can also cause significant morbidity and mortality on infection of the eye or brain and with disseminated infection of an immunosuppressed patient or a neonate. HSV growth inhibition measurement by plaque or yield reduction is a key task in the development of novel antiviral compounds but the manual methods are very labour intensive. The sensitive and specific PCR technology could be an effective method for quantitation of HSV DNA related to virus replication; however the currently described PCR approaches have a major limitation, namely the requirement of purification of DNA from the infected cells. This limitation makes this approach unfeasible for high-throughput screenings. The monitoring of HSV specific antibody titre is essential in vaccination trials and in the improvement of HSV-based oncolytic virotherapy. Usually, conventional cytopathic effect-based and plaque reduction neutralization tests are applied to measure the neutralization titre, but these methods are also time-consuming. To overcome this, we developed a quantitative PCR (qPCR) method for the detection of HSV-2 DNA directly from the infected cells (direct qPCR) and the method was further adapted to measure the titre of HSV specific neutralizing antibody in human sera. The conditions of direct qPCR assay were optimized to measure the antiviral activity of known and novel antiviral substances. Using HSV-2 seronegative and seropositive patients' sera, the validity of the direct qPCR neutralization test was compared to traditional cytopathic effect-based assay. The direct qPCR method was able to detect the HSV-2 DNA quantitatively between multiplicity of infection 1/64 and 1/4194304, indicating that the dynamic range of the detection was approximately 65,500 fold with high correlation between the biological and technical replicates. As a proof of the adaptability of the method, we applied the direct qPCR for antiviral inhibitory concentration 50 (IC50) measurements of known and novel antiviral compounds. The measured IC50 of acyclovir was ∼0.28µg/ml, similar to the previously published IC50 value. The IC50 of novel antiviral candidates was between 1.6-3.1µg/ml. The direct qPCR-based neutralization titres of HSV positive sera were 1:32-1:64, identical to the neutralization titres determined using a traditional neutralization assay. The negative sera did not inhibit the HSV-2 replication in either of the tests. Our direct qPCR method for the HSV-2 growth determination of antiviral IC50 and neutralization titre is less time-consuming, less subjective and a more accurate alternative to the traditional plaque titration and growth reduction assays.

Organotypic three-dimensional cancer cell cultures mirror drug responses in vivo: lessons learned from the inhibition of EGFR signaling.

Complex three-dimensional (3D) in vitro models that recapitulate human tumor biology are essential to understand the pathophysiology of the disease and to aid in the discovery of novel anti-cancer therapies. 3D organotypic cultures exhibit intercellular communication, nutrient and oxygen gradients, and cell polarity that is lacking in two-dimensional (2D) monolayer cultures. In the present study, we demonstrate that 2D and 3D cancer models exhibit different drug sensitivities towards both targeted inhibitors of EGFR signaling and broad acting cytotoxic agents. Changes in the kinase activities of ErbB family members and differential expression of apoptosis- and survival-associated genes before and after drug treatment may account for the differential drug sensitivities. Importantly, EGFR oncoprotein addiction was evident only in the 3D cultures mirroring the effect of EGFR inhibition in the clinic. Furthermore, targeted drug efficacy was strongly increased when incorporating cancer-associated fibroblasts into the 3D cultures. Taken together, we provide conclusive evidence that complex 3D cultures are more predictive of the clinical outcome than their 2D counterparts. In the future, 3D cultures will be instrumental for understanding the mode of action of drugs, identifying genotype-drug response relationships and developing patient-specific and personalized cancer treatments.