Biomarkers and biosensors for the diagnosis of noncompliant pH, dark cutting beef predisposition, and welfare in cattle.

Meat quality can be affected by stress, exhaustion, feed composition, and other physical and environmental conditions. These stressors can alter the pH in postmortem muscle, leading to high pH and low-quality dark cutting (DC) beef, resulting in considerable economic loss. Moreover, the dark cutting prediction may equally provide a measure for animal welfare since it is directly related to animal stress. There are two needs to advance on-site detection of dark cutters: (1) a clear indication that biomarker (signature compounds) levels in cattle correlate with stress and DC outcome; and (2) measuring these biomarkers rapidly and accurately on-farm or the abattoir, depending on the objectives. This critical review assesses which small molecules and proteins have been identified as potential biomarkers of stress and dark cutting in cattle. We discuss the potential of promising small molecule biomarkers, including catecholamine/cortisol metabolites, lactate, succinate, inosine, glucose, and ß-hydroxybutyrate, and we identify a clear research gap for proteomic biomarker discovery in live cattle. We also explore the potential of chemical-sensing and biosensing technologies, including direct electrochemical detection improved through nanotechnology (e.g., carbon and gold nanostructures), surface-enhanced Raman spectroscopy in combination with chemometrics, and commercial hand-held devices for small molecule detection. No current strategy exists to rapidly detect predictive meat quality biomarkers due to the need to further validate biomarkers and the fact that different biosensor types are needed to optimally detect different molecules. Nonetheless, several biomarker/biosensor combinations reported herein show excellent potential to enable the measurement of DC potential in live cattle.

The Requirement of Genetic Diagnostic Technologies for Environmental Surveillance of Antimicrobial Resistance.

Antimicrobial resistance (AMR) is threatening modern medicine. While the primary cost of AMR is paid in the healthcare domain, the agricultural and environmental domains are also reservoirs of resistant microorganisms and hence perpetual sources of AMR infections in humans. Consequently, the World Health Organisation and other international agencies are calling for surveillance of AMR in all three domains to guide intervention and risk reduction strategies. Technologies for detecting AMR that have been developed for healthcare settings are not immediately transferable to environmental and agricultural settings, and limited dialogue between the domains has hampered opportunities for cross-fertilisation to develop modified or new technologies. In this feature, we discuss the limitations of currently available AMR sensing technologies used in the clinic for sensing in other environments, and what is required to overcome these limitations.

Resonance Energy Transfer-Based Biosensors for Point-of-Need Diagnosis-Progress and Perspectives.

The demand for point-of-need (PON) diagnostics for clinical and other applications is continuing to grow. Much of this demand is currently serviced by biosensors, which combine a bioanalytical sensing element with a transducing device that reports results to the user. Ideally, such devices are easy to use and do not require special skills of the end user. Application-dependent, PON devices may need to be capable of measuring low levels of analytes very rapidly, and it is often helpful if they are also portable. To date, only two transduction modalities, colorimetric lateral flow immunoassays (LFIs) and electrochemical assays, fully meet these requirements and have been widely adopted at the point-of-need. These modalities are either non-quantitative (LFIs) or highly analyte-specific (electrochemical glucose meters), therefore requiring considerable modification if they are to be co-opted for measuring other biomarkers. Förster Resonance Energy Transfer (RET)-based biosensors incorporate a quantitative and highly versatile transduction modality that has been extensively used in biomedical research laboratories. RET-biosensors have not yet been applied at the point-of-need despite its advantages over other established techniques. In this review, we explore and discuss recent developments in the translation of RET-biosensors for PON diagnoses, including their potential benefits and drawbacks.

Structures of the transcriptional regulator BgaR, a lactose sensor.

The structure of BgaR, a transcriptional regulator of the lactose operon in Clostridium perfringens, has been solved by SAD phasing using a mercury derivative. BgaR is an exquisite sensor of lactose, with a binding affinity in the low-micromolar range. This sensor and regulator has been captured bound to lactose and to lactulose as well as in a nominal apo form, and was compared with AraC, another saccharide-binding transcriptional regulator. It is shown that the saccharides bind in the N-terminal region of a jelly-roll fold, but that part of the saccharide is exposed to bulk solvent. This differs from the classical AraC saccharide-binding site, which is mostly sequestered from the bulk solvent. The structures of BgaR bound to lactose and to lactulose highlight how specific and nonspecific interactions lead to a higher binding affinity of BgaR for lactose compared with lactulose. Moreover, solving multiple structures of BgaR in different space groups, both bound to saccharides and unbound, verified that the dimer interface along a C-terminal helix is similar to the dimer interface observed in AraC.

Highly Sensitive and Selective Biosensor for a Disaccharide Based on an AraC-Like Transcriptional Regulator Transduced with Bioluminescence Resonance Energy Transfer.

Sensitive and selective quantification of individual sugars in complex media is technically challenging and usually requires HPLC separation. Accurate measurement without the need for separation would be highly desirable. The measurement of trace levels of lactose in lactose-reduced milk exemplifies the problem, with the added challenge that trace lactose must be measured in the presence of ≈140 mM glucose and galactose, the products of lactase digestion of lactose. Biosensing is an alternative to HPLC, but current biosensing methods, based on coupled-enzyme assays, tend to have poor sensitivity and complex biochemistry and can be time-consuming. We explored a fundamentally different approach, based on identifying a lactose-specific binding protein compatible with photonic transduction. We identified the BgaR transcriptional regulator of Clostridium perfringens, which is highly selective for lactose, as a suitable ligand binding domain and combined it with a bioluminescence energy resonance transfer transduction system. This BRET-based biosensor showed a 27% decrease in the BRET ratio in the presence of saturating (1 mM) lactose. Using a 5 min assay, the half maximal effective concentration (EC50) for lactose in phosphate-buffered saline (PBS) was 12 µM. The biosensor was 200 times more sensitive to lactose than to glucose or galactose. Sensitivity and selectivity were not significantly affected by the presence of 10% (v/v) dialyzed milk. The biosensor is suitable for direct determination of residual lactose in lactase-treated milk, with a limit of detection of 0.2 µM, 100 times below the most stringent lactose-free standard and without the need to remove fat or protein from the sample.

ATP Recycling with Cell Lysate for Enzyme-Catalyzed Chemical Synthesis, Protein Expression and PCR.

E. coli lysate efficiently catalyzes acetyl phosphate-driven ATP regeneration in several important biotechnological applications. The utility of this ATP recycling strategy in enzyme-catalyzed chemical synthesis is illustrated through the conversion of uridine to UMP by the lysate from recombinant overexpression of uridine kinase with the E. coli. The UMP is further transformed into UTP through sequential phosphorylations by kinases naturally present in the lysate, in high yield. Cytidine and 5-fluorouridine also give the corresponding NMPs and NTPs with this system. Cell-free protein expression with a processed extract of lysate also proceeds readily when, instead of adding the required NTPs, all four are produced in situ from the NMPs, using acetyl phosphate and relying on endogenous kinase activity. Similarly, dNMPs can be used to produce the dNTPs necessary for DNA synthesis in PCR. These cheap alternative protocols showcase the potential of acetyl phosphate and ATP recycling with readily available cell lysate.

Peptide Synthesis through Cell-Free Expression of Fusion Proteins Incorporating Modified Amino Acids as Latent Cleavage Sites for Peptide Release.

Chlorinated analogues of Leu and Ile are incorporated during cell-free expression of peptides fused to protein, by exploiting the promiscuity of the natural biosynthetic machinery. They then act as sites for clean and efficient release of the peptides simply by brief heat treatment. Dehydro analogues of Leu and Ile are similarly incorporated as latent sites for peptide release through treatment with iodine under cold conditions. These protocols complement enzyme-catalyzed methods and have been used to prepare calcitonin, gastrin-releasing peptide, cholecystokinin-7, and prolactin-releasing peptide prohormones, as well as analogues substituted with unusual amino acids, thus illustrating their practical utility as alternatives to more traditional chemical peptide synthesis.

Dramatic increase of quench efficiency in "spacerless" dimaleimide fluorogens.

In this post-genomic era, new techniques are needed to cope with the task of assigning functional roles to the huge number of identified putative gene products. We have developed a minimalist labelling strategy based on the use of synthetic fluorogenic probe reagents that fluoresce only after their reaction with a target peptide sequence. The probe reagents have fluorescent cores and bear two maleimide groups, such that their latent fluorescence is quenched by a photoinduced electron transfer (PET) to the pendant maleimide groups, until both of these groups undergo a specific thiol addition reaction. The efficiency of the fluorescence quenching is critical to the practicality of this labelling method, and has been predicted to be related to the intramolecular distance between the fluorophore and the maleimide groups. We have conducted the first direct test of this hypothesis by preparing a series of novel fluorogens that differ only by the spacer moiety separating their coumarin fluorophore and their dimaleimide fragment. A striking correlation was observed between intramolecular distance and the fluorescence enhancement (FE) observed after reaction with two equivalents of thiol. Guided by this observation, we then designed 'spacerless' fluorogens, of which a dansyl derivative shows an FE ratio of >300, the largest recorded for dimaleimide fluorogens. The trends observed herein provide valuable lessons for subsequent fluorogen design, and the novel fluorogens developed in the course of this study are currently being applied to protein labelling applications.

De novo helical peptides as target sequences for a specific, fluorogenic protein labelling strategy.

New methods are needed to selectively label proteins in a manner that minimally perturbs their structures and functions. We have developed a 'small molecule'-based labelling technique that relies on the use of dimaleimide fluorogens that react with a target peptide sequence that presents appropriately spaced, solvent-exposed Cys residues. The thiol addition reaction between target sequence and dimaleimide fluorogen restores the latent fluorescence of the latter and results in the covalent fluorescent labelling of the protein of interest (J. Guy, K. Caron, S. Dufresne, S. W. Michnick, W. G. Skene and J. W. Keillor, J. Am. Chem. Soc., 2007, 129, 11969-11977). We demonstrated the proof-of-principle of this method previously, using a dicysteine mutant of the helical protein Fos (S. Girouard, M.-H. Houle, A. Grandbois, J. W. Keillor and S. W. Michnick, J. Am. Chem. Soc., 2005, 127, 559-566). Herein, we present the design of a novel peptide sequence presenting two Cys residues separated by two turns of an alpha-helix. The secondary structure of this sequence was confirmed by CD spectroscopy, before and after the fluorescent labelling reaction. A new series of di(3-methylmaleimide) fluorogens was prepared and kinetically evaluated, tuning their reactivity toward the target sequence. Attempts were made to increase the reactivity of the parent target sequence by rational design; however, the introduction of basic His residues in the vicinity of one or more Cys residues did not have the desired effect. Finally, epidermal growth factor receptors bearing the de novo target sequence were specifically labelled with a di(3-methylmaleimide) fluorescein fluorogen, validating our method for specific cell-surface labelling of proteins. A wide variety of fluorogen and peptide designs can be envisioned with potential applications to multiplexed labelling for the study of temporal and spatial dynamics of protein expression.

Convergent preparation and photophysical characterization of dimaleimide dansyl fluorogens: elucidation of the maleimide fluorescence quenching mechanism.

Dimaleimide fluorogens are being developed for application to fluorescent protein labeling. In this method, fluorophores bearing two maleimide quenching groups do not fluoresce until both maleimide groups have undergone thiol addition reactions with the Cys residues of the target protein sequence [J. Am. Chem. Soc. 2005, 127, 559-566]. In this work, a new convergent synthetic route was developed that would allow any fluorophore to be attached via a linker to a dimaleimide moiety in a modular fashion. Series of dimaleimide and dansyl derivatives were thus prepared conveniently and used to elucidate the mechanism of maleimide quenching. Intersystem crossing was ruled out as a potential quenching pathway, based on the absence of a detectable triplet intermediate by laser flash photolysis. Stern-Volmer rate constants were measured with exogenous dimaleimide quenchers and found to be close to the diffusion-controlled limits, consistent with electron transfer being thermodynamically favorable. The thermodynamic feasibility of the photoinduced electron transfer (PET) quenching mechanism was verified by cyclic voltammetry. The redox potentials measured for dansyl and maleimide confirm that electron transfer from the dansyl excited state to a pendant maleimide group is exergonic and is responsible for fluorescence quenching of the fluorogens studied herein. Taking this PET quenching mechanism into account, future fluorogenic protein labeling agents will be designed with spacers of variable length and rigidity to probe the structure-property PET efficiency relationship.

Synthesis and evaluation of peptidic maleimides as transglutaminase inhibitors.

A series of novel transglutaminase inhibitors was prepared, based on the scaffold of a commonly used peptide substrate and bearing an electrophilic maleimide group. These compounds were evaluated in vitro and shown to lead to irreversible inactivation of tissue transglutaminase. Comparison with inhibitors studied previously provides insight into the steric environment of the enzyme active site.