Pattern recognition of neuron specific enolase and carcinoembryonic antigen in whole blood samples

New tools and methods for pattern recognition of neuron specific enolase (NSE) and carcinoembryonic antigen (CEA) were proposed for the screening of whole blood samples. The new tools were based on stochastic sensors designed using nanoporous gold microspheres, graphite, graphene, diamond paste as well as α‐CDs, and 5,10,15,20‐tetraphenyl‐21H,23H‐porphyrin. The best sensor for the assay of CEA was the one based on P/graphite (the limit of determination was 16 fg/ml and sensitivity was 2.32 × 10⁷ s mg^?1 ml), while for the assay of NSE the, best sensor was the one based on P/graphene (the limit of determination was 7.45 pg/ml and sensitivity was 2.49 × 10⁸ s mg^?1 ml). The sensor of choice for simultaneous detection of NSE and CEA is the one based on P/graphene because we need high sensitivity and low limit of determination for NSE. To our knowledge, this is the only one screening test for early detection of lung cancer, by identification of NSE and CEA in whole blood samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Fast Screening of Whole Blood Samples and Pharmaceutical Compounds for Enantiorecognition of Free L‐T3, L‐T4, and D‐T4

A fast screening method of whole blood was proposed for enantiorecognition of free L‐T₃ , L‐T₄, and D‐T₄. Stochastic microsensors based on four inulins (IN, IQ, TEX, and HD) immobilized on diamond paste (DP) were used for recognition of free L‐T₃ , L‐T₄, and D‐T₄. For the enantiorecognition of free L‐T₄ and D‐T₄ in whole blood and pharmaceutical samples, the best microsensor was the one based on TEX/DP (wide linear concentration ranges, and low limits of quantification). The best limit of detection for the assay of free L‐T₃ (400 fmol/L) was recorded using the microsensors based on HD/DP, while for the assay of free L‐T4, and D‐T4 the best limit of determination (1 pmol/L) was recorded using the TX/DP‐based microsensor. For the enantiorecognition of free L‐T₃ in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification – of pmol/L magnitude order). For the enantiorecognition of free L‐T₃ in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification – of pmol/L magnitude order). Free L‐T₃ , L‐T₄, and D‐T₄ were recovered with high reliabilities in whole blood samples (recoveries higher than 99.00%, with RSD values lower than 1.00%) and pharmaceutical samples (recoveries higher than 95.00% with RSD values lower than 1.00%). Chirality 27:973–978, 2015. © 2015 Wiley Periodicals, Inc.     

Automated docking studies provide insights into molecular determinants of ligand recognition by N‐acetyl‐1‐d‐myo‐inosityl‐2‐amino‐2‐deoxy‐α‐d‐glucopyranoside deacetylase (MshB)

The metal‐dependent deacetylase N‐acetyl‐1‐d ‐myo‐inosityl‐2‐amino‐2‐deoxy‐α‐d ‐glucopyranoside deacetylase (MshB) catalyzes the deacetylation of N‐acetyl‐1‐d ‐myo‐inosityl‐2‐amino‐2‐deoxy‐α‐d ‐glucopyranoside (GlcNAc‐Ins), the committed step in mycothiol (MSH) biosynthesis. MSH is the thiol redox buffer used by mycobacteria to protect against oxidative damage and is involved in the detoxification of xenobiotics. As such, MshB is a target for the discovery of new drugs to treat tuberculosis (TB). While MshB substrate specificity and inhibitor activity have been probed extensively using enzyme kinetics, information regarding the molecular basis for the observed differences in substrate specificity and inhibitor activity is lacking. Herein we begin to examine the molecular determinants of MshB substrate specificity using automated docking studies with a set of known MshB substrates. Results from these studies offer insights into molecular recognition by MshB via identification of side chains and dynamic loops that may play roles in ligand binding. Additionally, results from these studies suggest that a hydrophobic cavity adjacent to the active site may be one important determinant of MshB substrate specificity. Importantly, this hydrophobic cavity may be advantageous for the design of MshB inhibitors with high affinity and specificity as potential TB drugs. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 406–417, 2014.     

Multivariate analysis of single quadrupole LC‐MS spectra for routine characterization and quantification of intact proteins

Modern high‐throughput proteomic platforms allow incomparable protein mixture resolution and identification. However, such sophisticated facilities are expensive and not always accessible for routine analysis of simple mixtures. In this paper, we propose a simple methodology, based on detection of intact, nondigested proteins by LC coupled to single quadrupole MS (sqLC‐MS), followed by the analysis of the resulting spectra by multivariate analysis (MA). By doing so, even large molecular weight (MW) proteins, generating complex spectra, can be characterized to a level that allows isoform discrimination, while standard algorithms, such as MS spectrum deconvolution, cannot. To demonstrate the effectiveness of the proposed approach, we have analyzed the spectra of a set of purified, intact albumins from seven different organisms (bovine, human, rabbit, rat, sheep, mouse, and pig) as a model of microheterogenous proteins, using Projection to Latent Structure Discriminant Analysis (PLS‐DA). Although these proteins are very similar (less than 1% difference in MW), sqLC‐MS/MA allowed their classification, and the identification of unknown source samples. In addition, MA allowed precise protein quantification from the same data (calibration curve R² = 0.9966). The ability to rapidly characterize and quantify proteins, together with simplicity and affordability, could make of combined sqLC‐MS/MA a routine method for the characterization of simple mixture of known proteins.     

Design of cyclic and d‐amino acids containing peptidomimetics for inhibition of protein‐protein interactions of HER2‐HER3

HER2 receptors are surface proteins belonging to the epidermal growth factor family of receptors. Their numbers are elevated in breast, lung, and ovarian cancers. HER2‐positive cancers are aggressive, have higher mortality rate, and have a poor prognosis. We have designed peptidomimetics that bind to HER2 and block the HER2‐mediated dimerization of epidermal growth factor family of receptors. Among these, a symmetrical cyclic peptidomimetic (compound 18 ) exhibited antiproliferative activity in HER2‐overexpressing lung cancer cell lines with IC₅₀ values in the nanomolar concentration range. To improve the stability of the peptidomimetic, d ‐amino acids were introduced into the peptidomimetic, and several analogs of compound 18 were designed. Among the analogs of compound 18 , compound 32 , a cyclic, d ‐amino acid‐containing peptidomimetic, was found to have an IC₅₀ value in the nanomolar range in HER2‐overexpressing cancer cell lines. The antiproliferative activity of compound 32 was also measured by using a 3D cell culture model that mimics the in vivo conditions. The binding of compound 32 to the HER2 protein was studied by surface plasmon resonance. In vitro stability studies indicated that compound 32 was stable in serum for 48 hours and intact peptide was detectable in vivo for 12 hours. Results from our studies indicated that 1 of the d ‐amino acid analogs of 18 , compound 32 , binds to the HER2 extracellular domain, inhibiting the phosphorylation of kinase of HER2.     

Ligand‐based pharmacophore modelling and screening of DNA minor groove binders targeting Staphylococcus aureus

The recognition of DNA by small molecules is of special importance in the design of new drugs. Many natural and synthetic compounds have the ability to interact with the minor groove of DNA. In the present study, identification of minor groove binding compounds was attained by the combined approach of pharmacophore modelling, virtual screening and molecular dynamics approach. Experimentally reported 32 minor groove binding compounds were used to develop the pharmacophore model. Based on the fitness score, best three pharmacophore hypotheses were selected and used as template for screening the compounds from drug bank database. This pharmacophore‐based screening provides many compounds with the same pharmacological properties. All these compounds were subjected to four phases of docking protocols with combined Glide‐quantum‐polarized ligand docking approach. Molecular dynamics results indicated that selected compounds are more active and showed good interaction in the binding site of DNA. Based on the scoring parameters and energy values, the best compounds were selected, and antibacterial activity of these compounds was identified using in vitro antimicrobial techniques. Copyright © 2014 John Wiley & Sons, Ltd.     

High‐throughput cell quantification assays for use in cell purification development – enabling technologies for cell production

High‐throughput screening (HTS) technology is gaining increasing importance in downstream process development of cell‐based products. The development of such HTS‐technologies, however, is highly dependent on the availability of robust, accurate, and sensitive high‐throughput cell quantification methods. In this article, we compare state‐of‐the‐art cell quantification methods with focus on their applicability in HTS‐platforms for downstream processing of cell‐based products. Sensitivity, dynamic range, and precision were evaluated for four methods that differ in their respective mechanism. In addition, we evaluated the performance of these methods over a range of buffer compositions, medium densities, and viscosities, representing conditions found in many downstream processing methods. We found that CellTiter‐Glo? and flow cytometry are excellent tools for high‐throughput cell quantification. Both methods have broad working ranges (3–4 log) and performed well over a wide range of buffer compositions. In comparison, CyQuant® Direct and CellTracker? had smaller working ranges and were more sensitive to changes in buffer composition. For fast and sensitive quantification of a single cell type, CellTiter‐Glo? performed best, while for more complex cell mixtures flow cytometry is the method of choice. Our analysis will facilitate the selection of the most suitable method for a specific application and provides a benchmark for future HTS development in downstream processing of cell‐based products.     

DeepQuanTR: MALDI‐MS‐based label‐free quantification of proteins in complex biological samples

The quantification of changes in protein abundance in complex biological specimens is essential for proteomic studies in basic and applied research. Here we report on the development and validation of the DeepQuanTR software for identification and quantification of differentially expressed proteins using LC‐MALDI‐MS. Following enzymatic digestion, HPLC peptide separation and normalization of MALDI‐MS signal intensities to the ones of internal standards, the software extracts peptide features, adjusts differences in HPLC retention times and performs a relative quantification of features. The annotation of multiple peptides to the corresponding parent protein allows the definition of a Protein Quant Value, which is related to protein abundance and which allows inter‐sample comparisons. The performance of DeepQuanTR was evaluated by analyzing 24 samples deriving from human serum spiked with different amounts of four proteins and eight complex samples of vascular proteins, derived from surgically resected human kidneys with cancer following ex vivo perfusion with a reactive ester biotin derivative. The identification and experimental validation of proteins, which were differentially regulated in cancerous lesions as compared with normal kidney, was used to demonstrate the power of DeepQuanTR. This software, which can easily be used with established proteomic methodologies, facilitates the relative quantification of proteins derived from a wide variety of different samples.     

Identification and ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry characterization of biosurfactants,including a new surfactin,isolated from oil‐contaminated environments

Biosurfactant‐producing bacteria were isolated from samples collected in areas contaminated with crude oil. The isolates were screened for biosurfactant production using qualitative drop‐collapse test, oil‐spreading and emulsification assays, and measurement of their tensoactive properties. Five isolates tested positive for in the screening experiments and displayed decrease in the surface tension below 30 mN m^?1. The biosurfactants produced by these isolates were further investigated and their molecular identification revealed that they are bacteria related to the Bacillus genus. Additionally, the biosurfactants produced were chemically characterized via UHPLC‐HRMS experiments, indicating the production of surfactin homologues, including a new class of these molecules.     

A porphyrin‐based fluorescence method for zinc determination in commercial propolis extracts without sample pretreatment

The quantification of zinc in over‐the–counter drugs as commercial propolis extracts by molecular fluorescence technique using meso ‐tetrakis(4‐carboxyphenyl)porphyrin (H₂TCPP⁴) was developed for the first time. The calibration curve is linear from 6.60 to 100 nmol L^?1 of Zn²⁺. The detection and quantification limits were 6.22 nmol L^?1 and 19.0 nmol L^?1, respectively. The reproducibility and repeatability calculated as the percentage variation of slopes of seven calibration curves were 6.75% and 4.61%, respectively. Commercial propolis extract samples from four Brazilian states were analyzed and the results (0.329–0.797 mg/100 mL) obtained with this method are in good agreement with that obtained with the Atomic Absorption Spectroscopy (AAS) technique. The method is simple, fast, of low cost and allows the analysis of the samples without pretreatment. Moreover the major advantage is that Zn‐porphyrin complex presents fluorescent characteristic promoting the selectivity and sensitivity of the method.     

A combination of two antibodies recognizing non‐overlapping epitopes of HER2 induces kinase activity‐dependent internalization of HER2

The human epidermal growth factor receptor 2 (HER2/ErbB2) is overexpressed in a number of human cancers. HER2 is the preferred heterodimerization partner for other epidermal growth factor receptor (EGFR) family members and is considered to be resistant to endocytic down‐regulation, properties which both contribute to the high oncogenic potential of HER2. Antibodies targeting members of the EGFR family are powerful tools in cancer treatment and can function by blocking ligand binding, preventing receptor dimerization, inhibiting receptor activation and/or inducing receptor internalization and degradation. With respect to antibody‐induced endocytosis of HER2, various results are reported, and the effect seems to depend on the HER2 expression level and whether antibodies are given as individual antibodies or as mixtures of two or more. In this study, the effect of a mixture of two monoclonal antibodies against non‐overlapping epitopes of HER2 was investigated with respect to localization and stability of HER2. Individual antibodies had limited effect, but the combination of antibodies induced internalization and degradation of HER2 by multiple endocytic pathways. In addition, HER2 was phosphorylated and ubiquitinated upon incubation with the antibody combination, and the HER2 kinase activity was found to be instrumental in antibody‐induced HER2 down‐regulation.     

Chiral single‐walled carbon nanotubes as chiral selectors in multimode enantioselective sensors

Single‐walled carbon nanotube‐(7,6) chirality was used for the design of multimode enantioselective sensors using different carbon matrices such as graphene paste, graphite paste, and carbon nanopowder‐based paste. l ‐ and d ‐malic acids were used as model analytes. The responses of the multimode sensors were evaluated for potentiometric and differential pulse voltammetry (DPV) modes. When carbon nanopowder was used as matrix, the multimode sensor was enantioselective for d ‐malic acid in the concentration range 10^?3 to 10^?15 mol/L for the potentiometric mode and 10^?5 to 10^?8 mol/L for the DPV mode. The graphite paste‐based sensor was enantioselective for l ‐malic acid in the ranges: 10^?10 to 10^?13 for the potentiometric mode and 10^?4 to 10^?7 mol/L for the DPV mode. The sensors based on graphene and chiral single‐walled carbon nanotubes were enantioselective for d ‐malic acid, and a response was obtained only in the DPV mode. Accordingly, the matrix influenced both the enantioselectivity and the sensitivity of the measurements. The application of the sensors was for the enantioanalysis of malic acid in wines and apple juice samples. The proposed method is fast and reliable and allows the quantification of l ‐ and d ‐malic acids using electrochemical methods based on different principles, from the real samples after a buffering of the samples. The enantioanalysis of malic acid in wine and juice samples was performed with high recoveries (higher than 90.00%) and low relative standard deviation (RSD) (%) values (lower than 1.00%).     

Docking and molecular dynamics studies of new potential inhibitors of the human epidermal receptor 2

Compounds similar to lapatinib and gefitinib have been investigated as potential inhibitors of the intracellular receptor tyrosine kinase (RTK) domain of the human epidermal receptor 2 (HER2), which is a promising molecular target to the drug design of new chemotherapies for breast, lung, ovarian and colorectal cancers. In this study, we have searched potential HER2 inhibitors used for treatment of other illnesses such as hepatitis, bacterial infections and sexual impotence screened in the DrugBank. The compounds selected were subjected to virtual screening docking in order to evaluate the main interactions between them and the RTK domain of HER2. The selected compounds were investigated by flexible docking, molecular dynamics studies and ΔG _bind calculations. The results suggest that antrafenine, saprisartan, reserpine, irinotecan and udenafil are potential candidates to inhibit the RTK domain of HER2.     

SNARE protein analog‐mediated membrane fusion

Fusion of lipid membranes to form a single bilayer is an essential process for life and provides important biological functions including neurotransmitter release. Membrane fusion proteins facilitate approximation of interacting membranes to overcome the energy barrier. In case of synaptic transmission, proteins involved are known as soluble N‐ethylmaleimide‐sensitive‐factor attachment receptor (SNARE) proteins. The SNAREs from synaptic vesicles interact with the SNAREs from the target membrane to form a coiled‐coil bundle of four helices, thus pulling the membranes tightly together and initiating fusion. However, it remains unclear how these proteins function at molecular level. Natural systems are often too complex to obtain unambiguous results. Simple model systems mimicking natural proteins in synthetic lipid bilayers are powerful tools for obtaining insights into this essential biological process. An important advantage of such systems is their well‐defined composition, which can be systematically varied in order to fully understand events at molecular level. In this review, selected model systems are presented based upon specific interactions between recognition units embedded in separate lipid bilayers mimicking native SNARE protein‐mediated membrane fusion. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Development of novel real‐time TaqMan® PCR assays for the species and sex identification of otter (Lutra lutra) and their application to noninvasive genetic monitoring

Developing strategies to maintain biodiversity requires baseline information on the current status of each individual species. The development of genetic techniques and their application to noninvasively collected samples have the potential to yield information on the structure of elusive animal populations and so are important tools in conservation management. Using DNA isolated from faecal samples can be challenging owing to low quantity and quality. This study, however, presents the development of novel real‐time polymerase chain reaction assays using fluorescently labelled TaqMan^® MGB probes enabling species and sex identification of Eurasian otter (Lutra lutra) spraints (faeces). These assays can also be used in determining an optimum microsatellite panel and can be employed as cost‐saving screening tools for downstream genetic testing including microsatellite genotyping and haplotype analysis. The techniques are shown to work efficiently with L. lutra DNA isolated from tissue, hair, spraint, blood and anal jelly samples.     

ITMSQ: A software tool for N‐ and C‐terminal fragment ion pairs based isobaric tandem mass spectrometry quantification

Tandem MS (MS2) quantification using the series of N‐ and C‐terminal fragment ion pairs generated from isobaric‐labelled peptides was recently considered an accurate strategy in quantitative proteomics. However, the presence of multiplexed terminal fragment ion in MS2 spectra may reduce the efficiency of peptide identification, resulting in lower identification scores or even incorrect assignments. To address this issue, we developed a quantitative software tool, denoted isobaric tandem MS quantification (ITMSQ), to improve N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantification. A spectrum splitting module was designed to separate the MS2 spectra from different samples, increasing the accuracy of both identification and quantification. ITMSQ offers a convenient interface through which parameters can be changed along with the labelling method, and the result files and all of the intermediate files can be exported. We performed an analysis of in vivo terminal amino acid labelling labelled HeLa samples and found that the numbers of quantified proteins and peptides increased by 13.64 and 27.52% after spectrum splitting, respectively. In conclusion, ITMSQ provides an accurate and reliable quantitative solutionfor N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantitative methods.     

SOFT‐MI: A novel microfabrication technique integrating soft‐lithography and molecular imprinting for tissue engineering applications

An innovative approach has been employed for the realization of bioactive scaffolds able to mimic the in vivo cellular microenvironment for tissue engineering applications. This method is based on the combination of molecular imprinting and soft‐lithography technology to enhance cellular adhesion and to guide cell growth and proliferation due to presence of highly specific recognition sites of selected biomolecules on a well‐defined polymeric microstructure. In this article polymethylmethacrylate (PMMA) scaffolds have been realized by using poly(dimethylsiloxane) (PDMS) microstructured molds imprinted with FITC‐albumin and TRITC‐lectin. In addition gelatin, an adhesion protein, was employed for the molecular imprinting of polymeric scaffolds for cellular tests. The most innovative aspect of this research was the molecular imprinting of whole cells for the development of substrates able to enhance the cell adhesion processes. Biotechnol. Bioeng. 2010;106: 804–817. © 2010 Wiley Periodicals, Inc.     

Molecular enantiorecognition of l‐glucose and d‐glucose in whole blood samples

In the past years, enantioanalysis became very important for clinical analysis; biomarkers/substances of biomedical importance with chiral structure should be analyzed and their presence correlated with the specific disorder. Therefore, we developed a method for the assay of l ‐ and d ‐glucose, based on molecular recognition of l ‐ and d ‐glucose. While for d ‐glucose there are many methods to assess its quantity, the l ‐enantiomer is not routinely detected by standard methods. Two stochastic microsensors based on the immobilization of Copper(II)phthalocyanine and Ni(II)phthalocyanine, in natural diamond powder, were proposed for the enantioanalysis of glucose. The proposed methods proved to have high sensitivities and were able to be used for determination of concentrations as low as 2.5 pg mL^?1 for d ‐glucose and as low as 2.5 fg mL^?1 for l ‐glucose. The enatioanalysis was performed with good results in whole blood samples collected from diabetic patients.     

Molecular recognition and binding of beta‐lactamase II from Bacillus cereus with penicillin V and sulbactam by spectroscopic analysis in combination with docking simulation

The molecular recognition and binding interaction of beta‐lactamase II from Bacillus cereus (Bc II) with penicillin V (PV) and sulbactam (Sul) at 277 K were studied by spectroscopic analysis and molecular docking. The results showed that a non‐fluorescence static complex was separately formed between Bc II and two ligands, the molecular ratio of Bc II to PV or Sul was both 1:1 in the binding and the binding constants were 2.00 × 10⁶ and 3.98 × 10⁵ (L/mol), respectively. The negative free energy changes and apparent activation energies indicated that both the binding processes were spontaneous. Molecular docking showed that in the binding process, the whole Sul molecule entered into the binding pocket of Bc II while only part of the whole PV molecule entered into the pocket due to a long side chain, and electrostatic interactions were the major contribution to the binding processes. In addition, a weak conformational change of Bc II was also observed in the molecular recognition and binding process of Bc II with PV or Sul. This study may provide some valuable information for exploring the recognition and binding of proteins with ligands in the binding process and for the design of novel super‐antibiotics.