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Abstract Background There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM) crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR) and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART) occurs at a constant temperature and only requires a simple light detection and integration device. Results Loop mediated isothermal amplification (LAMP) shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART) for determination of genetically modified (GM) maize target DNA at low levels of contamination (0.1-5.0% GM) using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation. Conclusions LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading within a reaction must be controlled to ensure quantification at low target concentrations.

Background: Cirrhosis detection in primary care relies on low-performing biomarkers. Consequently, up to 75% of subjects with cirrhosis receive their first diagnosis with decompensation when causal treatments are less effective at preserving liver function. We investigated an unprecedented approach to cirrhosis detection based on dynamic breath testing. Methods: We enrolled 29 subjects with cirrhosis (Child-Pugh A and B), and 29 controls. All subjects fasted overnight. Breath samples were taken using Breath Biopsy ® before and at different time points after the administration of 100 mg limonene. Absolute limonene breath levels were measured using gas chromatography-mass spectrometry. Results: All subjects showed a >100-fold limonene spike in breath after administration compared to baseline. Limonene breath kinetics showed first-order decay in >90% of the participants, with higher bioavailability in the cirrhosis group. At the Youden index, baseline limonene levels showed classification performance with an area under the roc curve (AUROC) of 0.83 ± 0.012, sensitivity of 0.66 ± 0.09, and specificity of 0.83 ± 0.07. The best performing timepoint post-administration was 60 min, with an AUROC of 0.91, sensitivity of 0.83 ± 0.07, and specificity of 0.9 ± 0.06. In the cirrhosis group, limonene bioavailability showed a correlation with MELD and fibrosis indicators, and was associated with signs of portal hypertension. Conclusions: Dynamic limonene breath testing enhances diagnostic performance for cirrhosis compared to static testing. The correlation with disease severity suggests potential for monitoring therapeutic interventions. Given the non-invasive nature of breath collection, a dynamic limonene breath test could be implemented in primary care.

DNA comprises molecular information stored in genetic and epigenetic bases, both of which are vital to our understanding of biology. Most DNA sequencing approaches address either genetics or epigenetics and thus capture incomplete information. Methods widely used to detect epigenetic DNA bases fail to capture common C-to-T mutations or distinguish 5-methylcytosine from 5-hydroxymethylcytosine. We present a single base-resolution sequencing methodology that sequences complete genetics and the two most common cytosine modifications in a single workflow. DNA is copied and bases are enzymatically converted. Coupled decoding of bases across the original and copy strand provides a phased digital readout. Methods are demonstrated on human genomic DNA and cell-free DNA from a blood sample of a patient with cancer. The approach is accurate, requires low DNA input and has a simple workflow and analysis pipeline. Simultaneous, phased reading of genetic and epigenetic bases provides a more complete picture of the information stored in genomes and has applications throughout biomedicine., (© 2023. The Author(s).)

Background and Aims: The prevalence of chronic liver disease in adults exceeds 30% in some countries and there is significant interest in developing tests and treatments to help control disease progression and reduce healthcare burden. Breath is a rich sampling matrix that offers non-invasive solutions suitable for early-stage detection and disease monitoring. Having previously investigated targeted analysis of a single biomarker, here we investigated a multiparametric approach to breath testing that would provide more robust and reliable results for clinical use., Methods: To identify candidate biomarkers we compared 46 breath samples from cirrhosis patients and 42 from controls. Collection and analysis used Breath Biopsy OMNI™, maximizing signal and contrast to background to provide high confidence biomarker detection based upon gas chromatography mass spectrometry (GC-MS). Blank samples were also analyzed to provide detailed information on background volatile organic compounds (VOCs) levels., Results: A set of 29 breath VOCs differed significantly between cirrhosis and controls. A classification model based on these VOCs had an area under the curve (AUC) of 0.95±0.04 in cross-validated test sets. The seven best performing VOCs were sufficient to maximize classification performance. A subset of 11 VOCs was correlated with blood metrics of liver function (bilirubin, albumin, prothrombin time) and separated patients by cirrhosis severity using principal component analysis., Conclusions: A set of seven VOCs consisting of previously reported and novel candidates show promise as a panel for liver disease detection and monitoring, showing correlation to disease severity and serum biomarkers at late stage., Competing Interests: Giuseppe Ferrandino, Giovanna De Palo, Antonio Murgia, Owen Birch, Rob Smith, Ahmed Tawfike, Olga Gandelman, Max Allsworth, and Billy Boyle, are/were employees of Owlstone Medical Ltd., (© 2023 Authors.)

The gold standard method for chronic liver diseases diagnosis and staging remains liver biopsy, despite the spread of less invasive surrogate modalities based on imaging and blood biomarkers. Still, more than 50% of chronic liver disease cases are detected at later stages when patients exhibit episodes of liver decompensation. Breath analysis represents an attractive means for the development of non-invasive tests for several pathologies, including chronic liver diseases. In this perspective review, we summarize the main findings of studies that compared the breath of patients with chronic liver diseases against that of control subjects and found candidate biomarkers for a potential breath test. Interestingly, identified compounds with best classification performance are of exogenous origin and used as flavoring agents in food. Therefore, random dietary exposure of the general population to these compounds prevents the establishment of threshold levels for the identification of disease subjects. To overcome this limitation, we propose the exogenous volatile organic compounds (EVOCs) probe approach, where one or multiple of these flavoring agent(s) are administered at a standard dose and liver dysfunction associated with chronic liver diseases is evaluated as a washout of ingested compound(s). We report preliminary results in healthy subjects in support of the potential of the EVOC Probe approach.

Introduction: Liver cirrhosis and its complication - hepatocellular carcinoma (HCC) - have been associated with increased exhaled limonene. It is currently unclear whether this increase is more strongly associated with the presence of HCC or with the severity of liver dysfunction., Methods: We compared the exhaled breath of 40 controls, 32 cirrhotic patients, and 12 cirrhotic patients with HCC using the Breath Biopsy platform. Breath samples were analyzed by thermal desorption-gas chromatography-mass spectrometry. Limonene levels were compared between the groups and correlated to bilirubin, albumin, prothrombin time international normalized ratio, and alanine aminotransferase., Results: Breath limonene concentration was significantly elevated in subjects with cirrhosis-induced HCC (M: 82.1 ng/L, interquartile range [IQR]: 16.33-199.32 ng/L) and cirrhosis (M: 32.6 ng/L, IQR: 6.55-123.07 ng/L) compared with controls (M: 6.2 ng/L, IQR: 2.62-9.57 ng/L) (P value = 0.0005 and 0.0001, respectively) with no significant difference between 2 diseased groups (P value = 0.37). Levels of exhaled limonene correlated with serum bilirubin (R = 0.25, P value = 0.0016, r = 0.51), albumin (R = 0.58, P value = 5.3e-8, r = -0.76), and international normalized ratio (R = 0.29, P value = 0.0003, r = 0.51), but not with alanine aminotransferase (R = 0.01, P value = 0.36, r = 0.19)., Discussion: Exhaled limonene levels are primarily affected by the presence of cirrhosis through reduced liver functional capacity, as indicated by limonene correlation with blood metrics of impaired hepatic clearance and protein synthesis capacity, without further alterations observed in subjects with HCC. This suggests that exhaled limonene is a potential non-invasive marker of liver metabolic capacity (see Visual abstract, Supplementary Digital Content 1, http://links.lww.com/CTG/A388).

Isothermal nucleic acid amplifications (iNAATs) have become an important alternative to PCR for in vitro molecular diagnostics in all fields. Amongst iNAATs Loop-mediated amplification (LAMP) has gained much attention over the last decade because of the simplicity of hardware requirements. LAMP demonstrates performance equivalent to that of PCR, but its application has been limited by the challenging primer design. The design of six primers in LAMP requires a selection of eight priming sites with significant restrictions imposed on their respective positioning and orientation. In order to relieve primer design constraints we propose an alternative approach which uses Stem primers instead of Loop primers and demonstrate the application of STEM-LAMP in assaying for Clostridium difficile, Listeria monocytogenes and HIV. Stem primers used in LAMP in combination with loop-generating and displacement primers gave significant benefits in speed and sensitivity, similar to those offered by Loop primers, while offering additional options of forward and reverse orientations, multiplexing, use in conjunction with Loop primers or even omission of one or two displacement primers, where necessary. Stem primers represent a valuable alternative to Loop primers and an additional tool for IVD assay development by offering more choices for primer design at the same time increasing assay speed, sensitivity, and reproducibility.

Cytoplasmic ATP can be measured continuously in single cardiac myocytes by monitoring the luminescence from microinjected firefly luciferase. We show here that the signals are markedly influenced by changes in cytoplasmic pH, and the calibration of the signals as ATP concentration is markedly affected by cytoplasmic protein. Measurements with a pH-sensitive fluorescent dye show that intracellular pH (pHi) can be clamped at pH 7.08 by perfusing cells with a modified bicarbonate-buffered Krebs saline containing 92 mM NaHCO3 and equilibrated with 20% CO2. Calibration of the firefly luciferase signal in vitro in the presence of high concentrations of BSA (180 mg/ml), to simulate the cytoplasmic protein concentration, revealed a shift in Km (ATP) to 2 mM, from approx. 400 microM in the absence of albumin in an identical ionic milieu. Luciferase measurements in pH-clamped cells show that metabolically poisoned isolated rat ventricle cardiomyocytes enter rigor at a cytoplasmic ATP concentration of between 1 and 2 mM. As the cells shorten in rigor, a process that is complete in 30-40 s, the cytoplasmic ATP concentration falls simultaneously to a level of typically 20 microM. When cyanide is removed 10 min later, to simulate reoxygenation, the signal recovers over a period of 2-3 min to a level approx. 70% of the original in the healthy cell. These studies indicate that rigor-mediated depletion of cytoplasmic ATP in metabolically poisoned cardiomyocytes is considerably more extreme than hitherto indicated.

In order to improve calibration of firefly luciferase signals obtained by injecting the enzyme into single, isolated heart and liver cells we have investigated why the luminescence from cells is greatly depressed compared with in vitro (in mammalian ionic milieu) and why the decay of the intracellular signal is remarkably slow. We have shown that inorganic pyrophosphatase greatly depresses the signal in vitro and that micromolar concentrations of inorganic pyrophosphate, comparable with that in cytoplasm, reverse this inhibition and stabilize the signal, eliminating its decay. Higher concentrations of pyrophosphate depress the signal by inhibiting ATP-binding to luciferase. Luciferase-injected cells exposed to extracellular luciferin concentrations above about 100 mumol/l (corresponding to a cytoplasmic level of c. 5-10 mumol/l because of a transplasmalemmal gradient) show a gradual, irreversible loss of signal. We attribute this phenomenon (which is not seen in vitro) to the gradual accumulation of a luminescently inactive, irreversible, luciferase-oxyluciferin complex. At low luciferin levels this complex is prevented from forming by cytoplasmic pyrophosphate. Above c. 100 mumol/l extracellular luciferin, the pyrophosphate level in the cytoplasm fails to fully prevent the complex forming. In vitro this phenomenon does not occur because the luciferase concentrations and hence oxyluciferin levels are orders of magnitude lower than in cells injected with concentrated luciferase solutions, which have a cytoplasmic luciferase concentration of approximately 2-4 mumol/l.