Prognostic Value of Circulating Cell-Free DNA Concentration and Neutrophil-to-Lymphocyte Ratio in Patients with Pancreatic Ductal Adenocarcinoma: A Prospective Cohort Study.

Circulating cell-free DNA (ccfDNA) quantity correlates with the clinical characteristics and prognosis of various cancer types. We investigated whether ccfDNA levels and the neutrophil-to-lymphocyte ratio (NLR) have prognostic value in patients with pancreatic ductal adenocarcinoma (PDAC). Peripheral blood was collected from 82 patients with PDAC prior to any diagnostic procedure or the administration of chemotherapy. Plasma DNA was isolated, and ccfDNA concentration and NLR were determined. We found that ccfDNA levels were correlated with age and tumor burden. Moreover, higher values of NLR (≥3.31) were linked with worse overall survival (OS) (4 vs. 10 months; log rank p = 0.011), and an elevated ccfDNA concentration (≥25.79 ng/mL) was strongly associated with shorter OS (4 vs. 8 months; log rank p = 0.009). According to the results of the multivariable Cox regression analysis, the baseline concentration of ccfDNA was an independent prognostic factor for OS (HR 0.45, 95% CI 0.21-0.97, p = 0.041). Furthermore, the combination of ccfDNA levels with NLR greatly enhanced the prognostic accuracy of PDAC patients. Our study demonstrates that ccfDNA concentration and NLR are independent predictors of survival in PDAC. Subsequent studies should validate this combination as a prognostic indicator in PDAC patients and assess its utility for guiding therapeutic decisions.

Phytosiderophore pathway response in barley exposed to iron, zinc or copper starvation.

Efficient micronutrient acquisition is a critical factor in selecting micronutrient dense crops for human consumption. Enhanced exudation and re-uptake of metal chelators, so-called phytosiderophores, by roots of graminaceous plants has been implicated in efficient micronutrient acquisition. We compared PS biosynthesis and exudation as a response mechanism to either Fe, Zn or Cu starvation. Two barley (Hordeum vulgare L.) lines with contrasting micronutrient grain yields were grown hydroponically and PS exudation (LC-MS) and root gene expression (RNAseq) were determined after either Fe, Zn, or Cu starvation. The response strength of the PS pathway was micronutrient dependent and decreased in the order Fe > Zn > Cu deficiency. We observed a stronger expression of PS pathway genes and greater PS exudation in the barley line with large micronutrient grain yield suggesting that a highly expressed PS pathway might be an important trait involved in high micronutrient accumulation. In addition to several metal specific transporters, we also found that the expression of IRO2 and bHLH156 transcription factors was not only induced under Fe but also under Zn and Cu deficiency. Our study delivers important insights into the role of the PS pathway in the acquisition of different micronutrients.

Surveying the mugineic acid family: Ion mobility - quadrupole time-of-flight mass spectrometry (IM-QTOFMS) characterization and tandem mass spectrometry (LC-ESI-MS/MS) quantification of all eight naturally occurring phytosiderophores.

Phytosiderophores (PS) are root exudates released by grass species (Poaceae) that play a pivotal role in iron (Fe) plant nutrition. A direct determination of PS in biological samples is of paramount importance in understanding micronutrient acquisition mediated by PS. To date, eight plant-born PS have been identified; however, no analytical procedure is currently available to quantify all eight PS simultaneously with high analytical confidence. With access to the full set of PS standards for the first time, we report comprehensive methods to both fully characterize (IM-QTOFMS) and quantify (LC-ESI-MS/MS) all eight naturally occurring PS belonging to the mugineic acid family. The quantitative method was fully validated, yielding linear results for all eight analytes, and no unwanted interferences with soil and plant matrices were observed. LOD and LOQ values determined for each PS were below 11 and 35 nmol L-1, respectively. The method's precision under reproducibility conditions (intra- and inter-day) of measurement was less than 2.5% RSD for all analytes. Additionally, all PS were annotated with high-resolution mass spectrometric fragment spectra and further characterized via drift tube ion mobility-mass spectrometry. The collision cross-sections obtained for primary ion species yielded a valuable database for future research focused on in-depth PS studies. The new quantitative method was applied to analyse root exudates from Fe-controlled and deficient barley, oat, rye, and sorghum plants. All eight PS, including mugineic acid (MA), 3"-hydroxymugineic acid (HMA), 3"-epi-hydroxymugineic acid (epi-HMA), hydroxyavenic acid (HAVA), deoxymugineic acid (DMA), 3"-hydroxydeoxymugineic acid (HDMA), 3"-epi-hydroxydeoxymugineic acid (epi-HDMA) and avenic acid (AVA) were for the first time successfully identified and quantified in root exudates of various graminaceous plants using a single analytical procedure. These newly developed methods can be applied to studies aimed at improving crop yield and micronutrient grain content for food consumption via plant-based biofortification.

Endoscopic ultrasound-guided fine-needle aspiration pancreatic adenocarcinoma samples yield adequate DNA for next-generation sequencing: A cohort analysis.

BACKGROUND: Genetic tests are increasingly performed for the management of unresectable pancreatic cancer. For genotyping aimed samples current guidelines recommend using core specimens, although based on moderate quality evidence. However, in clinical practice among the endoscopic ultrasound (EUS) guided tissue acquisition methods, fine needle aspiration (FNA) is the most widely performed. AIM: To assess the adequacy for next generation sequencing (NGS) of the DNA yielded from EUS-FNA pancreatic adenocarcinoma (PDAC) samples. METHODS: Between November 2018 and December 2021, 105 patients with PDAC confirmed by EUS-FNA were included in the study at our tertiary gastroenterology center. Either 22 gauge (G) or 19G FNA needles were used. One pass was dedicated to DNA extraction. DNA concentration and purity (A260/280, A260/230) were assessed by spectrophotometry. We assessed the differences in DNA parameters according to needle size and tumor characteristics (size, location) and the adequacy of the extracted DNA for NGS (defined as A260/280 ≥ 1.7, and DNA yield: ≥ 10 ng for amplicon based NGS, ≥ 50 ng for whole exome sequencing [WES], ≥ 100 ng for whole genome sequencing [WGS]) by analysis of variance and t-test respectively. Moreover, we compared DNA purity parameters across the different DNA yield categories. RESULTS: Our cohort included 49% male patients, aged 67.02 ± 8.38 years. The 22G needle was used in 71% of the cases. The DNA parameters across our samples varied as follows: DNA yield: 1289 ng (inter quartile range: 534.75-3101), A260/280 = 1.85 (1.79-1.86), A260/230 = 2.2 (1.72-2.36). DNA yield was > 10 ng in all samples and > 100 ng in 93% of them (one sample < 50 ng). There were no significant differences in the concentration and A260/280 between samples by needle size. Needle size was the only independent predictor of A260/230 which was higher in the 22G samples (P = 0.038). NGS adequacy rate was 90% for 19G samples regardless of NGS type, and for 22G samples it reached 89% for WGS adequacy and 91% for WES and amplicon based NGS. Samples with DNA yield > 100 ng had significantly higher A260/280 (1.89 ± 0.32 vs 1.34 ± 0.42, P = 0.013). Tumor characteristics were not corelated with the DNA parameters. CONCLUSION: EUS-FNA PDAC samples yield DNA adequate for subsequent NGS. DNA amount was similar between 22G and 19G FNA needles. DNA purity parameters may vary indirectly with needle size.

A quick and simple spectrophotometric method to determine total carbon concentrations in root exudate samples of grass species.

Purpose: Root exudates are key components driving belowground interaction between plant, microbes and soil. High-end analytical approaches provide advanced insights into exudate metabolite diversity, however, the amount of total carbon (C) released by roots should always be determined as the most basic parameter when characterizing root exudation as it (i) provides quantitative information of C exuded into the surrounding soil and (ii) allows to relate the abundance of individual exudate compounds to total C released. Here we propose a simple and quick, spectrophotometry-based method to quantify total dissolved organic carbon (DOC) concentration in exudation samples that is based on measuring the absorption of a pre-filtered but otherwise untreated exudate sample at 260 nm (DOC260). Method: Exudate samples collected from different grass genotypes (Zea mays, Oryza sativa, Hordeum vulgare) grown in various experimental settings (soil, hydroponic) were analysed with the DOC260 assay and results were compared with C concentrations obtained by liquid TOC-analyser. Conclusion: We demonstrated that the DOC260 method allowed for quick and inexpensive measurements of total dissolved organic carbon concentrations in exudate samples from grass species grown under nutrient sufficient as well as under P deficient conditions. Interestingly, DOC260 failed to predict DOC concentrations in exudate samples from plants grown under Zn and Fe deficiency suggesting a strong shift in metabolite composition under micronutrient deficiency. Even though the applicability of the DOC260 method remains to be tested on exudate samples originating from dicots and plants exposed to other environmental stresses (e.g. pathogen attack, heavy metal stress, etc), it will help to increase our understanding of root exudation and related rhizosphere processes in the future.

The Association between TRP Channels Expression and Clinicopathological Characteristics of Patients with Pancreatic Adenocarcinoma.

Pancreatic adenocarcinoma (PDAC) has low survival rates worldwide due to its tendency to be detected late and its characteristic desmoplastic reaction, which slows the use of targeted therapies. As such, the discovery of new connections between genes and the clinicopathological parameters contribute to the search for new biomarkers or targets for therapy. Transient receptor potential (TRP) channels are promising tools for cancer therapy or markers for PDAC. Therefore, in this study, we selected several genes encoding TRP proteins previously reported in cellular models, namely, Transient Receptor Potential Cation Channel Subfamily V Member 6 (TRPV6), Transient receptor potential ankyrin 1 (TRPA1), and Transient receptor potential cation channel subfamily M (melastatin) member 8 (TRPM8), as well as the TRPM8 Channel Associated Factor 1 (TCAF1) and TRPM8 Channel Associated Factor 2 (TCAF2) proteins, as regulatory factors. We analyzed the expression levels of tumors from patients enrolled in public datasets and confirmed the results with a validation cohort of PDAC patients enrolled in the Clinical Institute Fundeni, Romania. We found significantly higher expression levels of TRPA1, TRPM8, and TCAF1/F2 in tumoral tissues compared to normal tissues, but lower expression levels of TRPV6, suggesting that TRP channels have either tumor-suppressive or oncogenic roles. The expression levels were correlated with the tumoral stages and are related to the genes involved in calcium homeostasis (Calbindin 1 or S100A4) or to proteins participating in metastasis (PTPN1). We conclude that the selected TRP proteins provide new insights in the search for targets and biomarkers needed for therapeutic strategies for PDAC treatment.