Development of a real-time fluorescence resonance energy transfer PCR to identify the main pathogenic Campylobacter spp.

A simple real-time fluorescence resonance energy transfer (FRET) PCR, targeting the gyrA gene outside the quinolone resistance-determining region, was developed to identify Campylobacter jejuni and Campylobacter coli. These species were distinguished easily, as the corresponding melting points showed a difference of 15 degrees C. A second assay using the same biprobe and PCR conditions, but different PCR primers, was also developed to identify the less frequently encountered Campylobacter fetus. These assays were applied to 807 Campylobacter isolates from clinical specimens. Compared to phenotypic identification tests, the FRET assay yielded the same results for all except three of the isolates. Analysis by standard PCR and 16S rDNA sequencing demonstrated that two of these isolates were hippurate-negative C. jejuni strains, resulting in an erroneous phenotypic identification, while the third was an isolate of C. coli that contained a gyrA gene typical of C. jejuni, resulting in misidentification by the FRET assay. The FRET assay identified more isolates than standard PCR, which failed to yield amplification products with c. 10% of isolates. It was concluded that the FRET assays were rapid, reliable, reproducible and relatively cost-efficient, as they require only one biprobe and can be performed directly on boiled isolates.     

Detection of DNA Sequence with Enhanced Sensitivity and Higher FRET Efficiency Using a Light-Emitting Polymer,Peptide Nucleic Acid Probe and Anionic Surfactant System

An improved strategy has been developed for detection of DNA sequence by using water-soluble cationic conjugated polymer (PFP)/single-strand (ss) DNA and peptide nucleic acid labeled with fluorescent dye (PNAC*), where an anionic surfactant (sodium dodecyl sulphate, SDS) system has been used to improve the sensitivity of the sensor. The method of detection is simple to use, fast and cost-effective. This method uses the phenomenon of Forester Resonance Energy Transfer (FRET). The detection sensitivity of the biosensor has been improved by about ten times by using the anionic surfactant. It is observed that the effect of surfactant is to increase the photoluminescence (PL) intensity of the PNAC* when the sequence of the DNA is complementary (to that of PNA probe). On the other hand when the two sequences are non-complementary, the PL intensity of the PNAC* is further reduced as compared to the case when surfactant was absent.     

Scintillation proximity assay in lead discovery

Background: Scintillation proximity assay (SPA) is a homogeneous scintillant bead-based platform for the measurement of biological processes and plays an important role in the identification of active chemical entities in drug discovery. Objective: The design and development of solid-phase SPA approaches are examined and compared with alternative non-radiometric fluorescence-based technologies. Methods: This review provides background on the principle of SPA and its application to biomolecular interactions from a variety of biological sources. Conclusion: The SPA approach is well suited to the demands of commercial high volume automation and assay miniaturization for target-based high-throughput screening campaigns on synthetic and natural product libraries as well as for benchtop characterization and confirmation studies. In the near future, innovations in the way SPA and fluorescence-based screening strategies are multiplexed will improve our comprehensive understanding of cellular system biology and dramatically advance the lead discovery process for the treatment of complex target-related disorders.     

Synthetic heparan sulfate standards and machine learning facilitate the development of solid-state nanopore analysis

The application of solid-state (SS) nanopore devices to single-molecule nucleic acid sequencing has been challenging. Thus, the early successes in applying SS nanopore devices to the more difficult class of biopolymer, glycosaminoglycans (GAGs), have been surprising, motivating us to examine the potential use of an SS nanopore to analyze synthetic heparan sulfate GAG chains of controlled composition and sequence prepared through a promising, recently developed chemoenzymatic route. A minimal representation of the nanopore data, using only signal magnitude and duration, revealed, by eye and image recognition algorithms, clear differences between the signals generated by four synthetic GAGs. By subsequent machine learning, it was possible to determine disaccharide and even monosaccharide composition of these four synthetic GAGs using as few as 500 events, corresponding to a zeptomole of sample. These data suggest that ultrasensitive GAG analysis may be possible using SS nanopore detection and well-characterized molecular training sets.

Glycosaminoglycans (GAGs) are linear anionic polysaccharides found on cell surfaces and in the extracellular matrix in all animals. GAGs comprise an important class of biopolymers that are ubiquitous in nature and exhibit a number of critical functional roles including biological recognition and signaling (1–3). Such processes play critical roles in physiology, such as in development and wound healing, and pathophysiology, such as cancer and infectious disease. Sulfated GAGs result from template-independent synthesis in the Golgi of animal cells (4, 5) and are polydisperse, heteropolysaccharides comprising variable disaccharide repeating units that are classified by these repeating units. Like nucleic acids, sulfated GAGs are made up of repeating units that comprise a linear sequence (Fig. 1). Unlike the nucleic acids, GAGs have far more complicated structures and number of possible sequences and they present severe challenges to both synthesis and characterization. Thus, we undertook to chemoenzymatically synthesize defined GAGs and characterize these using solid-state nanopore analysis.Open in a separate windowFig. 1.Structures of four synthetic GAG samples. Polysaccharide NSH is made up of N-sulfoglucosamine (GlcNS) and glucuronic acid (GlcA),NS2S is made up with GlcNS and 2-O-sulfo-iduronic acid (IdoA2S), NS6S is made up with 6-O-sulfo-N-sulfoheparosan (GlcNS6S) and GlcA, and NS6S2S is made up with GlcNS6S and IdoA2S.Despite their structural complexities, sulfated GAGs often contain well-defined domain structures that are responsible for their diverse biological functions, yet even this level of structural complexity poses a significant general challenge to structural analysis and sequencing. The simple, short-chain, chondroitin sulfate GAG component of bikunin has been sequenced using liquid chromatography–tandem mass spectrometry (LC-MS/MS) (6). While LC-MS/MS is capable of sequencing such simple, short-chain GAGs, it is not yet able to distinguish all of the many isobaric isomers of the variably sulfated saccharide residues and uronic acid epimers commonly encountered in more structurally complex GAGs, such as heparan sulfate (HS) (7). NMR has been applied to determine GAG structures but often requires milligram amounts of samples. HS/heparin is made up of →4)-β-d-glucuronic acid (GlcA) [or α-l-iduronic acid (IdoA)] (1→4)-α-d-glucosamine (GlcN) [1→ repeating units with 2-O-sulfo (S) groups on selected uronic acid residues and 3- and/or 6-O-S and N-S or N-acetyl (Ac) group substitutions on the glucosamine residues] (Fig. 1). GAG structural analysis presents challenges beyond their chemical complexity. There are no amplification methods to detect small numbers of GAG chains, whereas nucleic acid analysis can rely on PCR. Similarly, there are few GAG-specific antibodies or aptamers (8), and no natural GAG chromophores or fluorophores (9), in contrast to the many used for protein sensing. Ultrasensitive (zeptomole) detection methods of modified GAGs, based on fluorescence resonance energy transfer (FRET) (10), DNA bar coding (11), and dye-based nanosensors (12) have been demonstrated, but their application to sequencing is particularly challenging because of the high level of structural complexity of sulfated GAGs.Nanopore single-molecule detection is now routinely applied to DNA (13, 14) and RNA (15–17) biopolymers, and is increasingly applied to protein characterization (18–22). In brief, a nanopore is a nanofluidic channel ∼10 nm long and <100 nm in diameter, serving as the sole fluid connection between two reservoirs of electrolyte separated by an otherwise impermeable membrane (Fig. 2A). On applying a voltage across this nanopore, the passage of supporting electrolyte ions results in a “baseline,” or open-pore current, i₀. The passage of a biopolymer analyte through this nanopore disrupts the flow of supporting electrolyte ions, often as a current blockage. This temporary reduction in ionic current is called an “event,” and its magnitude (mean blockage ratio over the dwell time, ⟨f_b⟩=⟨i⟩_Td/⟨i₀⟩) and its temporal features [dwell time (Td)] (Fig. 2 B and C) depend on the size and shape of the nanopore, the biopolymer analyte, and the applied voltage and interfacial charge distributions. Indeed, the passage of DNA through engineered protein nanopore devices produces current blockages that can be applied in sequencing, and the widespread use of these commercial protein nanopore DNA sequencing devices is increasing (23, 24). Despite this success with protein nanopores, the potential benefits of (abiotic) solid-state (SS) nanopores have continued to drive development efforts. Such a transition to the freely size-tunable SS platform (25, 26), however, is vital for the application of nanopores to the characterization of branched glycans (27). Yet the use of SS nanopores in even the better-established DNA sensing regime remains challenging. The application of nanopore sensing to glycans, while promising, remains profoundly exploratory using nanopores of any kind. The transition to the SS nanopores is accompanied by significant changes in pore geometry, chemistry, characteristics, and potential analyte–pore interactions and sensing modalities, so that there is a critical need for studies in the realm of nanopore glycomics (27, 28). For example, outcomes of early nanopore studies on a structurally simple unsulfated GAG, hyaluronan (HA, →4)- β -GlcA (1 → 3)- β -GlcNAc (1→), while providing some information on HA size does not provide definitive structural information (29, 30). SS nanopore analysis of two sulfated GAGs, heparin and a heparin contaminant, oversulfated chondroitin sulfate, using a silicon nitride SS nanopore was able to qualitatively identify these GAGs by either the magnitude or duration of characteristic current blockages (28). SS nanopore data on GAGs, analyzed using a machine-learning (ML) algorithm (i.e., a support vector machine [SVM]), distinguished heparin and chondroitin sulfate oligosaccharides and unfractionated heparin and low molecular weight heparin with >90% accuracy (31).Open in a separate windowFig. 2.Nanopore characteristics of four samples. (A) Schematic of the nanopore configuration. Anionic GAGs driven by electrophoresis to and through the pore with a negative applied voltage would be detected if they perturbed the open-pore current. (B) A representative current trace and events from polysaccharide NS6S2S test using an ∼6-nm-diameter nanopore. Measurements were collected using a −150-mV applied-voltage (details in Results and Discussion, and Materials and Methods) (C) Scatter plots of dwell time vs. current blockage ratio for four polysaccharides. To remove the bias of event numbers in human image recognition, all plots contain only the first 2,475 events. (D) PCA visualization of the embedded images from the four unique GAGs. The blue circles and region represent NSH, the red X and region represents NS2S, the green triangle and region represents NS6S, and the brown cross and region represents NS6S2S. The algorithm clusters signals from each GAG based on scatter plot images. Each insert shows one 500-events image from each sample class. All 500-events images are in SI Appendix, Fig. S12.Nanopore studies on GAGs, and glycans more broadly, have been severely limited by the lack of a library of structurally defined standards. The uniformity of sulfated GAGs prepared from animal sources is difficult to control and exhibits significant sequence heterogeneity and polydispersity (32). HS is particularly problematic as even for a small HS hexasaccharide, composed of an IdoA/GlcA:GlcNS/GlcNAc sequence with 12 available sites for random sulfation, there are 32,768 possible sequences. Recently, chemoenzymatic synthesis has made inroads in the preparation of high-purity sulfated HS GAGs from heparosan (→4)- β -GlcA (1→4)- β -GlcNAc (1→) (33). HS GAGs of approximately the same chain length and polydispersity and having a single repeating disaccharide unit (SI Appendix, Table S1) including, NSH (→4)- β -GlcA (1→4)- β -GlcNS (1→), NS2S (→4)-α α -IdoA2S (1 → 4)- β -GlcNS (1→), NS6S (→4)- β -GlcA (1→4)- β -GlcNS6S(1→)), NS6S2S (→4)- α -IdoA2S (1→4)- β -GlcNS6S (1→) have been prepared (see Materials and Methods and ref. 34) (Fig. 1). Here we use our recently developed synthetic technique, which has proven difficult to benchmark, in conjunction with a nanopore technique, which has only just begun to be applied to glycomics and has been severely challenged by the lack of available high-quality samples, to develop a fully integrated approach for the nanopore analysis of complex carbohydrates.     

Bile salt-induced apoptosis involves NADPH oxidase isoform activation

BACKGROUND & AIMS: Hydrophobic bile salts trigger a rapid oxidative stress response as an upstream event of CD95 activation and hepatocyte apoptosis. METHODS: The underlying mechanisms were studied by Western blot, immunocytochemistry, protein knockdown, and fluorescence resonance energy transfer microscopy in rat hepatocytes and human hepatoma cell line 7 (Huh7). RESULTS: The rapid oxidative stress formation in response to taurolithocholate-3-sulfate (TLCS) was inhibited by diphenyleneiodonium, apocynin, and neopterin, suggestive for the involvement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. TLCS induced a rapid serine phosphorylation of the regulatory subunit p47phox, which was sensitive to inhibition of sphingomyelinase and protein kinase Czeta (PKCzeta). Inhibitors of p47phox phosphorylation and p47phox protein knockdown abolished the TLCS-induced oxidative stress response and blunted subsequent CD95 activation. Consequences of TLCS-induced oxidative stress were c-Jun-N-terminal kinase activation and Yes-dependent activation of the epidermal growth factor receptor (EGFR), followed by EGFR-catalyzed CD95 tyrosine phosphorylation, formation of the death-inducing signaling complex, and execution of apoptosis. As shown by fluorescence resonance energy transfer experiments in Huh7 cells, TLCS induced a c-Jun-N-terminal kinase-dependent EGFR/CD95 association in the cytosol and trafficking of this protein complex to the plasma membrane. Inhibition of EGFR tyrosine kinase activity by AG1478 allowed for cytosolic EGFR/CD95 association, but prevented targeting of the EGFR/CD95 complex to the plasma membrane. Both processes, and TLCS-induced Yes and EGFR activation, were sensitive to inhibition of sphingomyelinase, PKCzeta, or NADPH oxidases. CONCLUSIONS: The data suggest that hydrophobic bile salts activate NADPH oxidase isoforms with the resulting oxidative stress response triggering activation of the CD95 system and apoptosis.     

Förster resonance energy transfer (FRET)-Labeled nanoprobe enables real-time diagnosis of pancreatic juice activation due to postoperative pancreatic fistula

ObjectivesPostoperative pancreatic fistula (POPF) subsequent to pancreatectomy often causes activation of pancreatic juice, resulting in serious complications. In POPF, the types of pancreatic juices found are active and inactive, and the identification of these two types of pancreatic juice greatly contributes to the development of postoperative management after pancreatectomy. This study reports favorable results of the clinical application of the Förster resonance energy transfer (FRET) nanoprobe that was independently developed to distinguish between the active and inactive types of pancreatic juice.MethodsThe FRET nanoprobe developed was a nanoprotein capsule. It exuded a red color when the capsule structure was maintained. When activated protease in the pancreatic juice acts on it, the capsules are reduced quantitatively and FRET is abolished, resulting in a change in color from red to green. Pancreatic juice activation can be measured by the FRET signal. A total of 117 drainage fluid samples from 16 postpancreatoduodenectomy cases were obtained and evaluated.ResultsThe diagnosis of pancreatic juice activation was possible using the FRET signal with a cut-off value of 1.6. Pancreatic juice activation was not associated with drainage fluid amylase (AMY) levels. The results demonstrated that pancreatic juice was activated when drainage fluid was infected.ConclusionThe use of a FRET nanoprobe enabled real-time detection of the presence or absence of pancreatic juice activation in pancreatic fistula after pancreatic surgery. There was an adequate correlation between infection and pancreatic juice activation regardless of drain AMY levels.     

EGF receptor family: twisting targets for improved cancer therapies

Abstract

The epidermal growth factor receptor (EGFR) undergoes a conformational change in response to ligand binding. The ligand-induced changes in cell surface aggregation and mobility have a profound effect on the function of all the family members. Ligand also activates the EGFR intracellular kinase, stimulating proliferation and cell survival. The EGFR family are often activated, overexpressed or mutated in cancer cells and therapeutic drugs (including antibodies) can slow the progress of some cancers. This article provides a brief, annotated summary of the presentations and discussion which occurred at the Epidermal Growth Factor Receptor – Future Directions Conference held in Jerusalem in November 2013.     

A frameshifting stimulatory stem loop destabilizes the hybrid state and impedes ribosomal translocation

Ribosomal frameshifting occurs when a ribosome slips a few nucleotides on an mRNA and generates a new sequence of amino acids. Programmed −1 ribosomal frameshifting (−1PRF) is used in various systems to express two or more proteins from a single mRNA at precisely regulated levels. We used single-molecule fluorescence resonance energy transfer (smFRET) to study the dynamics of −1PRF in the Escherichia coli dnaX gene. The frameshifting mRNA (FSmRNA) contained the frameshifting signals: a Shine–Dalgarno sequence, a slippery sequence, and a downstream stem loop. The dynamics of ribosomal complexes translating through the slippery sequence were characterized using smFRET between the Cy3-labeled L1 stalk of the large ribosomal subunit and a Cy5-labeled tRNA^Lys in the ribosomal peptidyl-tRNA–binding (P) site. We observed significantly slower elongation factor G (EF-G)–catalyzed translocation through the slippery sequence of FSmRNA in comparison with an mRNA lacking the stem loop, ΔSL. Furthermore, the P-site tRNA/L1 stalk of FSmRNA-programmed pretranslocation (PRE) ribosomal complexes exhibited multiple fluctuations between the classical/open and hybrid/closed states, respectively, in the presence of EF-G before translocation, in contrast with ΔSL-programmed PRE complexes, which sampled the hybrid/closed state approximately once before undergoing translocation. Quantitative analysis showed that the stimulatory stem loop destabilizes the hybrid state and elevates the energy barriers corresponding to subsequent substeps of translocation. The shift of the FSmRNA-programmed PRE complex equilibrium toward the classical/open state and toward states that favor EF-G dissociation apparently allows the PRE complex to explore alternative translocation pathways such as −1PRF.The ribosome is the molecular machine that synthesizes proteins by translating messenger RNAs (mRNAs); each sequence of 3 nt, 1 codon, characterizes 1 aa (1–3). Failure to maintain frame during translation occurs with a low error of 10⁻⁵ (4); however, frameshifting with high efficiency (>10⁻²) is often programmed into many mRNAs to express two or more proteins from a single mRNA (5, 6). Many RNA viruses, including HIV-1, use programmed frameshifting to produce their vital proteins at a precise ratio (7, 8). The common −1 programmed ribosomal frameshifting (−1PRF) signals are a heptanucleotide slippery sequence (X XXY YYZ, underlining denotes the zero-frame) and a downstream stimulatory secondary structure such as a stem loop or a pseudoknot. Frameshifting that takes place on the slippery sequence results in minimal base pair mismatches. Prokaryotic systems have an additional stimulatory signal, an upstream, internal Shine–Dalgarno (SD) sequence (9). The dnaX gene of Escherichia coli has the three −1PRF signals; an SD sequence, an A AAA AAG slippery sequence, and a downstream stem loop (9–12). Highly efficient (50–80%) −1PRF during translation of the mRNA results in production of the γ DNA-polymerase subunit in the −1 frame and the τ DNA-polymerase subunit in the 0 frame (10).The −1PRF signals are spaced so that the slippery sequence is positioned within the ribosomal peptidyl-tRNA–binding (P) site and aminoacyl-tRNA–binding (A) site, whereas the downstream secondary structure is positioned at the ribosomal mRNA entry channel (Fig. 1) (5–8, 13). The upstream SD sequence base pairs with 16S ribosomal RNA (rRNA) near the ribosomal tRNA exit (E) site (Fig. 1) (9). Both the SD sequence and the downstream secondary structure can cause pausing during translation (14–19). However, frameshifting efficiency is not strictly related to the pausing extent (15, 17), and it is not proportional to the thermodynamic or mechanical stabilities of the secondary structures (7, 20). Nonetheless, it does correlate with the thermodynamic stability of the first 3–4 bp of the downstream secondary structure (21), and with the conformational plasticity of this structure (7, 20). However, a mechanism by which the stimulatory secondary structure promotes efficient frameshifitng has not emerged yet.Open in a separate windowFig. 1.A programmed −1 FSmRNA construct and a schematic drawing of a ribosomal complex translating the slippery sequence. FSmRNA contains three −1PRF signals from the dnaX gene in E. coli; an SD sequence, a slippery sequence, and a downstream stem loop. ΔSL mRNA has the same sequence as FSmRNA except with the stem loop (red box) deleted. Start and stop codons are highlighted in blue. Corresponding polypeptide sequences are shown below the mRNA. A schematic drawing of the POST-(Cy5)K₁ complex shows the 50S and 30S subunits in blue and purple rectangles, respectively. The L1 stalk in the small blue rectangle is labeled with Cy3. The ribosomal complex contains fMVK-(Cy5)tRNA^Lys in the P site, where the slippery sequence is being displayed. The upstream SD sequence forms base pairs with 16S rRNA and the downstream stem loop presents at the mRNA entry channel in the 30S subunit. The orange oval denotes the biotin on a DNA primer annealed to the 5′ end of the mRNA for immobilization.A translational elongation cycle starts with selecting a correct aminoacyl-tRNA in the A site via conformational changes of the posttranslocation (POST) ribosomal complex that are triggered upon binding an EF-Tu(GTP)⋅aminoacyl-tRNA ternary complex (TC) (1). Once peptidyl transfer takes place, the resulting pretranslocation (PRE) ribosomal complex undergoes large-scale conformational changes that facilitate translocation of the tRNAs from the P and A sites into the E and P sites, simultaneously advancing the ribosome along the mRNA by 3 nt (22). In the first step of translocation, the acceptor stems of the tRNAs are repositioned within the large ribosomal (50S, in prokaryotes) subunit to move the tRNAs from their classical (P/P, A/A) state to their hybrid (P/E, A/P) states, where X and Y in the X/Y notation refer to the position of the anticodon stem loop (ASL) of the tRNA in the small ribosomal (30S, in prokaryotes) subunit and the position of the acceptor stem of the tRNA in the 50S subunit, respectively. Hybrid state (H) formation is accompanied by rotation of the 30S subunit relative to the 50S subunit (23, 24) and a closure of the L1 stalk of the 50S subunit such that it forms a direct contact with the P/E hybrid tRNA (23–25), a global conformation of the PRE complex that we refer to as “global state 2” (25). Global state 1, in contrast, contains classical state (C) tRNAs, nonrotated subunits, and an open L1 stalk (25). Single-molecule fluorescence resonance energy transfer (smFRET) studies of this step of translocation have shown that the H state forms spontaneously upon peptidyl transfer and that, in the absence of an elongation factor-G (EF-G), the H state exists in a dynamic equilibrium with the C state (25–27). Translocation is completed by movement of the ASLs of the tRNAs and the mRNA in the 30S subunit. This step, which comprises the rate-limiting step for the overall process of translocation, requires unlocking of the PRE complex, a conformational change that is thought to involve swiveling of the head domain of the 30S subunit (28, 29) and that is catalyzed by EF-G (30). smFRET and structural studies suggest that the L1 stalk–P/E hybrid tRNA interaction that is established during the first step of translocation is preserved throughout the second step of translocation and is essential for guiding the translocation of the P/E hybrid tRNA into the E site (25, 31, 32).Here, we report an smFRET study of the dynamics of ribosomal complexes programmed with the −1PRF mRNA of the E. coli dnaX gene. We used a FRET pair composed of a Cy3-labeled L1 stalk [L1(Cy3)-stalk] and a Cy5-labeled P-site tRNA^Lys [(Cy5)tRNA^Lys] on the first lysine codon in the slippery sequence. As previously demonstrated (25), this FRET pair enabled us to monitor transitions of ribosomal complexes between C and H states and the subsequent release of the translocated (Cy5)tRNA^Lys from the E site, along one round of the translational elongation cycle. Two mRNA constructs, one containing the downstream stem loop and one lacking it, were compared to study the effect of the secondary structure on the dynamics and translocation of the ribosomal complexes. Our results show that the downstream stem loop changes the dynamics of the PRE ribosomal complexes and disturbs the translocation process. We propose that frameshifting is one of the favorable paths that the ribosome can adopt during the futile EF-G–driven translocation attempts from the H state.