1H NMR-based metabolomics analysis of human saliva, other oral fluids, and/or tissue biopsies serves as a valuable technique for the exploration of metabolic processes, and when associated with ’state-of-the-art’ multivariate (MV) statistical analysis strategies, provides a powerful means of examining the identification of characteristic metabolite patterns, which may serve to differentiate between patients with oral health conditions (e.g., periodontitis, dental caries, and oral cancers) and age-matched heathy controls. This approach may also be employed to explore such discriminatory signatures in the salivary 1H NMR profiles of patients with systemic diseases, and to date, these have included diabetes, Sjörgen’s syndrome, cancers, neurological conditions such as Alzheimer’s disease, and viral infections. However, such investigations are complicated in view of quite a large number of serious inconsistencies between the different studies performed by independent research groups globally; these include differing protocols and routes for saliva sample collection (e.g., stimulated versus unstimulated samples), their timings (particularly the oral activity abstention period involved, which may range from one to 12 h or more), and methods for sample transport, storage, and preparation for NMR analysis, not to mention a very wide variety of demographic variables that may influence salivary metabolite concentrations, notably the age, gender, ethnic origin, salivary flow-rate, lifestyles, diets, and smoking status of participant donors, together with their exposure to any other possible convoluting environmental factors. In view of the explosive increase in reported salivary metabolomics investigations, in this update, we critically review a wide range of critical considerations for the successful performance of such experiments. These include the nature, composite sources, and biomolecular status of human saliva samples; the merits of these samples as media for the screening of disease biomarkers, notably their facile, unsupervised collection; and the different classes of such metabolomics investigations possible. Also encompassed is an account of the history of NMR-based salivary metabolomics; our recommended regimens for the collection, transport, and storage of saliva samples, along with their preparation for NMR analysis; frequently employed pulse sequences for the NMR analysis of these samples; the supreme resonance assignment benefits offered by homo- and heteronuclear two-dimensional NMR techniques; deliberations regarding salivary biomolecule quantification approaches employed for such studies, including the preprocessing and bucketing of multianalyte salivary NMR spectra, and the normalization, transformation, and scaling of datasets therefrom; salivary phenotype analysis, featuring the segregation of a range of different metabolites into ‘pools’ grouped according to their potential physiological sources; and lastly, future prospects afforded by the applications of LF benchtop NMR spectrometers for direct evaluations of the oral or systemic health status of patients at clinical ‘point-of-contact’ sites, e.g., dental surgeries. This commentary is then concluded with appropriate recommendations for the conduct of future salivary metabolomics studies. Also included are two original case studies featuring investigations of (1) the 1H NMR resonance line-widths of selected biomolecules and their possible dependence on biomacromolecular binding equilibria, and (2) the combined univariate (UV) and MV analysis of saliva specimens collected from a large group of healthy control participants in order to potentially delineate the possible origins of biomolecules therein, particularly host- versus oral microbiome-derived sources. In a follow-up publication, Part II of this series, we conduct censorious reviews of reported observations acquired from a diversity of salivary metabolomics investigations performed to evaluate both localized oral and non-oral diseases. Perplexing problems encountered with these again include those arising from sample collection and preparation protocols, along with 1H NMR spectral misassignments.

Summary Although poly-ADP-ribosylation (PARylation) of DNA repair factors had been well documented, its role in the repair of DNA double-strand breaks (DSBs) is poorly understood. NR4A nuclear orphan receptors were previously linked to DSB repair; however, their function in the process remains elusive. Classically, NR4As function as transcription factors using a specialized tandem zinc-finger DNA-binding domain (DBD) for target gene induction. Here, we show that NR4A DBD is bi-functional and can bind poly-ADP-ribose (PAR) through a pocket localized in the second zinc finger. Separation-of-function mutants demonstrate that NR4A PAR binding, while dispensable for transcriptional activity, facilitates repair of radiation-induced DNA double-strand breaks in G1. Moreover, we define DNA-PKcs protein as a prominent target of ionizing radiation-induced PARylation. Mechanistically, NR4As function by directly targeting poly-ADP-ribosylated DNA-PKcs to facilitate its autophosphorylation-promoting DNA-PK kinase assembly at DNA lesions. Selective targeting of the PAR-binding pocket of NR4A presents an opportunity for cancer therapy.

Human saliva offers many advantages over other biofluids regarding its use and value as a bioanalytical medium for the identification and prognostic monitoring of human diseases, mainly because its collection is largely non-invasive, is relatively cheap, and does not require any major clinical supervision, nor supervisory input. Indeed, participants donating this biofluid for such purposes, including the identification, validation and quantification of surrogate biomarkers, may easily self-collect such samples in their homes following the provision of full collection details to them by researchers. In this report, the authors have focused on the applications of metabolomics technologies to the diagnosis and progressive severity monitoring of human cancer conditions, firstly oral cancers (e.g., oral cavity squamous cell carcinoma), and secondly extra-oral (systemic) cancers such as lung, breast and prostate cancers. For each publication reviewed, the authors provide a detailed evaluation and critical appraisal of the experimental design, sample size, ease of sample collection (usually but not exclusively as whole mouth saliva (WMS)), their transport, length of storage and preparation for analysis. Moreover, recommended protocols for the optimisation of NMR pulse sequences for analysis, along with the application of methods and techniques for verifying and resonance assignments and validating the quantification of biomolecules responsible, are critically considered. In view of the authors’ specialisms and research interests, the majority of these investigations were conducted using NMR-based metabolomics techniques. The extension of these studies to determinations of metabolic pathways which have been pathologically disturbed in these diseases is also assessed here and reviewed. Where available, data for the monitoring of patients’ responses to chemotherapeutic treatments, and in one case, radiotherapy, are also evaluated herein. Additionally, a novel case study featured evaluates the molecular nature, levels and diagnostic potential of 1H NMR-detectable salivary ‘acute-phase’ glycoprotein carbohydrate side chains, and/or their monomeric saccharide derivatives, as biomarkers for cancer and inflammatory conditions.

Ammonia (NH3) has been shown to be a key biomarker for a wide variety of diseases, such as hepatic and chronic kidney diseases (CKD), and cancers. It also has relevance to the oral health research area, and, hence, its determination in appropriate biofluids and tissues is of much importance. However, since it contains exchangeable >N-H protons, its analysis via 1H NMR spectroscopy, which is a widely employed technique in untargeted metabolomic studies, is rendered complicated. In this study, we focused on the 1H NMR analysis of this biomarker in less invasively collected human saliva samples, and we successfully identified and quantified it as ammonium cation (NH4+) in post-collection acidulated forms of this biofluid using both the standard calibration curve and standard addition method (SAM) approaches. For this purpose, n = 27 whole mouth saliva (WMS) samples were provided by healthy human participants, and all donors were required to follow a fasting/oral environment abstention period of 8 h prior to collection. Following acidification (pH 2.00), diluted WMS supernatant samples treated with 10% (v/v) D2O underwent 1H NMR analysis (600 MHz). The acquired results demonstrated that NH4+ can be reliably determined in these supernatants via integration of the central line of its characteristic 1:1:1 intensity triplet resonance (complete spectral range δ = 6.97–7.21 ppm). Experiments performed also demonstrated that any urease-catalysed NH3 generation occurring post-sampling in WMS samples did not affect the results acquired during the usual timespan of laboratory processing required prior to analysis. Further experiments demonstrated that oral mouth-rinsing episodes conducted prior to sample collection, as reported in previous studies, gave rise to major decreases in salivary NH4+ levels thereafter, which renormalised to only 50–60% of their basal control concentrations at the 180-minute post-rinsing time point. Therefore, the WMS sample collection method employed significantly affected the absolute levels of this analyte. The LLOD was 60 μmol/L with 128 scans. The mean ± SD salivary NH4+ concentration of WMS supernatants was 11.4 ± 4.5 mmol/L. The potential extension of these analytical strategies to the screening of other metabolites with exchangeable 1H nuclei is discussed, as is their relevance to the monitoring of human disorders involving the excessive generation and/or uptake of cellular/tissue material, or altered homeostasis, in NH3.

This communication represents Part III of our series of reports based on the applications of human saliva as a useful and conveniently collectable medium for the discovery, identification and monitoring of biomarkers, which are of some merit for the diagnosis of human diseases. Such biomarkers, or others reflecting the dysfunction of specific disease-associated metabolic pathways, may also be employed for the prognostic pathological tracking of these diseases. Part I of this series set the experimental and logistical groundwork for this report, and the preceding paper, Part II, featured the applications of newly developed metabolomics technologies to the diagnosis and severity grading of human cancer conditions, both oral and systemic. Clearly, there are many benefits, both scientific and economic, associated with the donation of human saliva samples (usually as whole mouth saliva) from humans consenting to and participating in investigations focused on the discovery of biomolecular markers of diseases. These include usually non-invasive collection protocols, relatively low cost when compared against blood sample collection, and no requirement for clinical supervision during collection episodes. This paper is centred on the employment and value of ‘state-of-the-art’ metabolomics technologies to the diagnosis and prognosis of a wide range of non-cancerous human diseases. Firstly, these include common oral diseases such as periodontal diseases (from type 1 (gingivitis) to type 4 (advanced periodontitis)), and dental caries. Secondly, a wide range of extra-oral (systemic) conditions are covered, most notably diabetes types 1 and 2, cardiovascular and neurological diseases, and Sjögren’s syndrome, along with a series of viral infections, e.g., pharyngitis, influenza, HIV and COVID-19. Since the authors’ major research interests lie in the area of the principles and applications of NMR-linked metabolomics techniques, many, but not all, of the studies reviewed were conducted using these technologies, with special attention being given to recommended protocols for their operation and management, for example, satisfactory experimental model designs; sample collection and laboratory processing techniques; the selection of sample-specific NMR pulse sequences for saliva analysis; and strategies available for the confirmation of resonance assignments for both endogenous and exogenous molecules in this biofluid. This article also features an original case study, which is focussed on the use of NMR-based salivary metabolomics techniques to provide some key biomarkers for the diagnosis of pharyngitis, and an example of how to ‘police’ such studies and to recognise participants who perceive that they actually have this disorder but do not from their metabolic profiles and multivariate analysis pattern-based clusterings. The biochemical and clinical significance of these multidimensional metabolomics investigations are discussed in detail.

The BCL2L1 gene expresses two isoforms of Bcl-x protein via the use of either of two alternative 5′ splice sites (5′ss) in exon 2. These proteins have antagonistic actions, Bcl-XL being anti-apoptotic and Bcl-XS pro-apoptotic. In a number of cancers the Bcl-XL isoform is over-expressed, resulting in cancer cell survival and growth, so switching splicing to the Xs isoform could have therapeutic benefits. We have previously proposed that a putative G-quadruplex (G4) exists downstream of the XS 5′ss and shown that the ellipticine derivative GQC-05, a previously identified DNA G4-specific ligand, induces an increase in the XS/XL ratio both in vitro and in cells. Here, we demonstrate that this G4 forms in vitro and that the structure is stabilised in the presence of GQC-05. We also show that GQC-05 binds RNA non-specifically in buffer conditions, but selectively to the Bcl-x G4 in the presence of nuclear extract, highlighting the limitations of biophysical measurements taken outside of a functional environment. We also demonstrate that GQC-05 is able to shift the equilibrium between competing G4 and duplex structures towards the G4 conformation, leading to an increase in accessibility of the XS 5′ss, supporting our previous model on the mechanism of action of GQC-05.