Evaluation of a commercially developed semiautomated PCR-surface-enhanced raman scattering assay for diagnosis of invasive fungal disease.

Nonculture-based tests are gaining popularity in the diagnosis of invasive fungal disease (IFD), but PCR is excluded from disease-defining criteria because of limited standardization and a lack of commercial assays. Commercial PCR assays may have a standardized methodology while providing quality assurance. The detection of PCR products by a surface-enhanced Raman scattering (SERS) assay potentially provides superior analytical sensitivity and multiplexing capacity compared to that of real-time PCR. Using this approach, the RenDx Fungiplex assay was developed to detect Candida and Aspergillus. Analytical and clinical evaluations of the assay were undertaken using extraction methods according to European Aspergillus PCR Initiative (EAPCRI) recommendations. A total of 195 previously extracted samples (133 plasma, 49 serum, and 13 whole blood) from 112 patients (29 with proven/probable IFD) were tested. The 95% limit of detection of Candida and Aspergillus was 200 copies per reaction, with an overall reproducibility of 92.1% for detecting 20 input copies per PCR, and 89.8% for the nucleic acid extraction-PCR-SERS process for detecting fungal burdens of <20 genome equivalents per sample. A clinical evaluation showed that assay positivity significantly correlated with IFD (P < 0.0001). The sensitivity of the assay was 82.8% and was similar for both Candida (80.0%) and Aspergillus (85.7%). The specificity was 87.5% and was increased (97.5%) by using a multiple (≥ 2 samples) PCR-positive threshold. In summary, the RenDx Fungiplex assay is a PCR-SERS assay for diagnosing IFD and demonstrates promising clinical performance on a variety of samples. This was a retrospective clinical evaluation, and performance is likely to be enhanced through a prospective analysis of clinical validity and by determining clinical utility.

Analysis of intracellular enzyme activity by surface enhanced Raman scattering.

Dysfunctional intracellular enzymatic activity is believed to be an underlying cause of a myriad of diseases. We present the first use of surface enhanced Raman scattering (SERS) as a detection technique capable of reporting intracellular activity of a specific enzyme. Careful choice of reagents allowed the preparation of high resolution cellular activity maps highlighting the specific conversion of the commonly used ELISA reagent 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside (X-Gal), by wild type ß-galactosidase enzymes. Further, through co-addition of X-Gal substrate and inhibitors we were able to demonstrate that intracellular substrate conversion occurred predominantly through an enzymatically specific pathway. The data presented therefore supports the application of SERS probes as sensitive, specific sensors of biochemical activity and demonstrates the use of SERS probes for the first time as beacons capable of high resolution subcellular localisation of native enzymes.

Characterizing wine terroir using strontium isotope ratios: a review.

This paper presents a detailed review of the use of 87Sr/86Sr isotope systematics for wine provenance studies. The method is based on the principle that the Sr isotope ratio in wine reflects that of the labile fraction of the vineyard soil from which the wine is produced. The review encompasses 87Sr/86Sr data from wine samples published between 1993 and 2021 from terroirs in 22 different countries. The analytical procedures and techniques adopted by the different authors and the range of isotope ratios obtained in the different studies are discussed and evaluated. This study provides a bibliometric analysis of the 87Sr/86Sr isotope approach for wine authentication at different scales. Although limitations are evident when implemented at large (global) scales, we demonstrate that the 87Sr/86Sr isotope tracing technique remains a powerful and reliable tool for determining the geographical origin of wine when combined with detailed knowledge of the geological and soil characteristics of the substrata. For example, this combination of data allows the wines grown in the volcanic soils of Central and Southern Italy to be unambiguously fingerprinted. We present a detailed protocol for the application of the Sr isotope technique to wine authentication.

Detection of inflammation in vivo by surface-enhanced Raman scattering provides higher sensitivity than conventional fluorescence imaging.

The detection of inflammatory changes is a key aim for the early diagnosis and treatment of several autoimmune, infectious, and metastatic diseases. While surface-enhanced Raman scattering (SERS) has the capability to provide noninvasive, in vivo imaging at sufficient depth to achieve this goal, this approach has not been exploited in the study of inflammation. SERS-active nanoparticles were coded with a unique Raman signal that was protected under a wide range of conditions and stimuli. To detect early-stage inflammation, gold nanoparticle clusters containing Raman-active molecules were conjugated to intercellular adhesion molecule 1- (ICAM-1-) specific monoclonal antibodies. SERS allowed noninvasive measurement of ICAM-1 expression in vivo with twice the sensitivity of two-photon fluorescence. This is the first time SERS has been used for in vivo detection of inflammation and is a major advance in the ever-growing toolkit of approaches for use in noninvasive, next-generation in vivo imaging.

Tracking bisphosphonates through a 20 mm thick porcine tissue by using surface-enhanced spatially offset Raman spectroscopy.

Track it down: A recognized surface-enhanced Raman scattering (SERS) nanotag signal was monitored from a thin, dispersed layer of bisphosphonate-functionalized nanotags on a bone sample, through a 20 mm thick specimen of porcine muscle tissue by surface-enhanced spatial offset Raman spectroscopy (SESORS; see picture). The result demonstrates the great potential for non-invasive in vivo bisphosphonate drug tracking.

A platform for oncogenomic reporting and interpretation.

Manual interpretation of variants remains rate limiting in precision oncology. The increasing scale and complexity of molecular data generated from comprehensive sequencing of cancer samples requires advanced interpretative platforms as precision oncology expands beyond individual patients to entire populations. To address this unmet need, we introduce a Platform for Oncogenomic Reporting and Interpretation (PORI), comprising an analytic framework that facilitates the interpretation and reporting of somatic variants in cancer. PORI integrates reporting and graph knowledge base tools combined with support for manual curation at the reporting stage. PORI represents an open-source platform alternative to commercial reporting solutions suitable for comprehensive genomic data sets in precision oncology. We demonstrate the utility of PORI by matching 9,961 pan-cancer genome atlas tumours to the graph knowledge base, calculating therapeutically informative alterations, and making available reports describing select individual samples.

Nanoparticles and inflammation.

The development of nanoscale molecular probes capable of diagnosis, characterization, and clinical treatment of disease is leading to a new generation of imaging technologies. Such probes are particularly relevant to inflammation, where the detection of subclinical, early disease states could facilitate speedier detection that could yield enhanced, tailored therapies. Nanoparticles offer robust platforms capable of sensitive detection, and early research has indicated their suitability for the detection of vascular activation and cellular recruitment at subclinical levels. This suggests that nanoparticle techniques may provide excellent biomarkers for the diagnosis and progression of inflammatory diseases with magnetic resonance imaging (MRI), fluorescent quantum dots (QDs), and surface enhanced Raman scattering (SERS) probes being just some of the new methodologies employed. Development of these techniques could lead to a range of sensitive probes capable of ultrasensitive, localized detection of inflammation. This article will discuss the merits of each approach, with a general overview to their applicability in inflammatory diseases.

Functionalized nanoparticles for bioanalysis by SERRS.

Metallic nanoparticles can be used as basic materials for a wide variety of purposes including building blocks for nanoassemblies, substrates for enhanced spectroscopies such as fluorescence and Raman and as labels for biomolecules. In the present paper, we report how silver and gold nanoparticles can be functionalized with specific biomolecular probes to interact in a specific manner with a target molecule to provide a change in the properties of the nanoparticles which can be measured to indicate the molecular recognition event. Examples of this approach include DNA hybridization to switch on SERRS (surface-enhanced resonance Raman scattering) when a specific target sequence is present, the use of nanoparticles for in vivo SERRS imaging and the use of nanoparticles functionalized with antibodies to provide a new type of immunoassay. These examples indicate how nanoparticles can be used to provide highly sensitive and informative data from a variety of biological systems when used in combination with SERRS.

Quantitative SERRS immunoassay for the detection of human PSA.

We report the first use of a commonly used ELISA colorimetric substrate as a SERRS marker and show how it can be used for the detection of pg ml(-1) levels of human prostate specific antigen (PSA) in clinical samples. The technique is amenable over a wide range of concentrations and lends itself to future multiplexing analysis.

In situ detection of pterins by SERS.

Surface enhanced Raman scattering (SERS) has been used to detect specific pterin molecules at sub-nanomolar concentrations. SERS is fast becoming a widely used technique for the sensitive and specific detection of multiple analytes. The information-rich and concentration-dependent spectra obtained from SERS make the technique ideally placed for high speed, low cost analysis of almost any analyte. Further, to show the feasibility of SERS in the detection of biologically relevant targets, a synthetic pterin analogue of the naturally occurring pterin cofactor, tetrahydrobiopterin, has been detected at a series of concentrations and the method used for the successful detection of the synthetic pterin in mouse serum. In this analysis, spectroscopic collection was optimized for water-based pteridine derivatives using two visible wavelengths of excitation (514.5 and 632.8 nm) and differing mesoscopic metal nanoparticles allowing the limits of detection to be calculated.

Binding of 14-3-3 proteins to a single stranded oligodeoxynucleotide aptamer.

A synthetic library of ca. 10(13) single stranded oligodeoxynucleotides, each comprising a randomized 40mer sequence and homogeneous 10mer flanking regions, was screened for binding to recombinant human 14-3-3gamma. A single aptamer, which showed similar affinities (K(D) approximately 10(-8)M) for six isoforms of the protein, has been shown to bind to undenatured 14-3-3 protein in the cerebral spinal fluid of scrapie infected sheep.

Strontium isotope characterization of wines from Quebec, Canada.

The (87)Sr/(86)Sr isotope ratios were measured on grape, wine and soil samples collected in 13 commercial vineyards located in three major wine producing areas of Quebec (Canada). The soils yield Sr isotope ratios that are intimately related to the local geology and unambiguously discriminate the different producing areas. A strong relationship exists between the (87)Sr/(86)Sr isotope ratios of the wine and the grapes. This suggests that the vinification process does not alter the overall Sr budget. Although the Sr isotope ratios of the grapes do not show a strong correlation with the bulk Sr isotope composition of the soil, they do correlate strongly with the Sr isotope composition contained in the labile fraction of the soil. This indicates that the labile fraction of the soil represents the Sr reservoir available to the plant during its growth. This study demonstrates that the Sr isotope approach can be used as a viable tool in forensic science for investigating the provenance of commercial wines.

Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells.

INTRODUCTION: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. METHODS: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. FINDINGS: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. CONCLUSIONS: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein.

Combining functionalised nanoparticles and SERS for the detection of DNA relating to disease.

DNA functionalised nanoparticle probes offer new opportunities in analyte detection. Ultrasensitive, molecularly specific targeting of analytes is possible through the use of metallic nanoparticles and their ability to generate a surface enhanced Raman scattering (SERS) response. This is leading to a new range of diagnostic clinical probes based on SERS detection. Our approaches have shown how such probes can detect specific DNA sequences by using a biomolecular recognition event to 'turn on' a SERS response through a controlled assembly process of the DNA functionalised nanoparticles. Further, we have prepared DNA aptamer functionalised SERS probes and demonstrated how introduction of a protein target can change the aggregation state of the nanoparticles in a dose-dependant manner. These approaches are being used as methods to detect biomolecules that indicate a specific disease being present with a view to improving disease management.

Imaging inflammation in real time--future of nanoparticles.

The detection of subclinical early inflammation in autoimmune diseases is an important but currently technically demanding approach to direct initial diagnosis and subsequent choice of therapy. Recent advances in imaging using NP provides the potential to detect cellular recruitment, vascular activation or leakage at a subclinically stage of disease and may provide predictive "biomarkers" of future pathogenesis. The NP used are either untargeted and taken up by phagocytic cells, or are linked to a ligand, targeting localisation to the site of inflammation. Techniques, varying from MRI and fluorescence to Raman spectroscopy are being employed. In this short review, we summarise many of the recent developments in the field of NP imaging related to inflammation.

Neodymium-142 evidence for Hadean mafic crust.

Neodymium-142 data for rocks from the Nuvvuagittuq greenstone belt in northern Quebec, Canada, show that some rock types have lower 142Nd/144Nd ratios than the terrestrial standard (epsilon142Nd = -0.07 to -0.15). Within a mafic amphibolite unit, 142Nd/144Nd ratios correlate positively with Sm/Nd ratios and produce a 146Sm-142Nd isochron with an age of 4280(-81)(+53) million years. These rocks thus sample incompatible-element-enriched material formed shortly after Earth formation and may represent the oldest preserved crustal section on Earth.

Discovery of glutathione S-transferase inhibitors using dynamic combinatorial chemistry.

Protein-directed dynamic combinatorial chemistry (DCC) relies on reversible chemical reactions that can function under the near-physiological conditions required by the biological target. Few classes of reaction have so far proven effective at generating dynamic combinatorial libraries (DCLs) under such constraints. In this study, we establish the conjugate addition of thiols to enones as a reaction well-suited for the synthesis of dynamic combinatorial libraries (DCLs) directed by the active site of the enzyme glutathione S-transferase (GST). The reaction is fast, freely reversible at basic pH, and easily interfaced with the protein, which is a target for the design of inhibitors in cancer therapy and the treatment of parasitic diseases such as schistosomiasis. We have synthesized DCLs based on glutathione (GSH, 1) and the enone ethacrynic acid, 2a. By varying either set of components, we can choose to probe either the GSH binding region ("G site") or the adjacent hydrophobic acceptor binding region ("H site") of the GST active site. In both cases the strongest binding DCL components are identified due to molecular amplification by GST which, in the latter system, leads to the identification of two new inhibitors for the GST enzyme.