Evaluation of pre-analytical factors affecting plasma DNA analysis.

Pre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci. Using this assay, we compared 7 cfDNA extraction kits and found cfDNA yield and fragment size vary significantly. We also compared 3 blood collection protocols using plasma samples from 23 healthy volunteers (EDTA tubes processed within 1 hour and Cell-free DNA Blood Collection Tubes processed within 24 and 72 hours) and found no significant differences in cfDNA yield, fragment size and background noise between these protocols. In 219 clinical samples, cfDNA fragments were shorter in plasma samples processed immediately after venipuncture compared to archived samples, suggesting contribution of background DNA by lysed peripheral blood cells. In summary, we have described a multiplexed ddPCR assay to assess quality of cfDNA samples prior to downstream molecular analyses and we have evaluated potential sources of pre-analytical variation in cfDNA studies

Health-related quality of life and utilities in gastric premalignant conditions and malignant lesions: a multicentre study in a high prevalence country

BACKGROUND AND AIMS: A recent review of economic studies relating to gastric cancer revealed that authors use different tests to estimate utilities in patients with and without gastric cancer. Our aim was to determine the utilities of gastric premalignant conditions and adenocarcinoma with a single standardized health measure instrument. METHODS: Cross-sectional nationwide study of patients undergoing upper endoscopy (n=1,434) using the EQ-5D-5L quality of life (QoL) questionnaire. RESULTS: According to EQ-5D-5L, utilities in individuals without gastric lesions were 0.78 (95% confidence interval: 0.76-0.80), with gastric premalignant conditions 0.79 (0.77-0.81), previously treated for gastric cancer 0.77 (0.73-0.81) and with present cancer 0.68 (0.55-0.81). Self-reported QoL according to the visual analogue scale (VAS) for the same groups were 0.67 (0.66-0.69), 0.67 (0.66-0.69), 0.62 (0.59-0.65) and 0.62 (0.54-0.70) respectively. Utilities were consistently lower in women versus men (no lesions 0.71 vs. 0.78; premalignant conditions 0.70 vs. 0.82; treated for cancer 0.72 vs. 0.78 and present cancer 0.66 vs. 0.70). CONCLUSION: The health-related QoL utilities of patients with premalignant conditions are similar to those without gastric diseases whereas patients with present cancer show decreased utilities. Moreover, women had consistently lower utilities than men. These results confirm that the use of a single standardized instrument such as the EQ-5D-5L for all stages of the gastric carcinogenesis cascade is feasible and that it captures differences between conditions and gender dissimilarities, being relevant information for authors pretending to conduct further cost-utility analysis

Enzymatic amide bond formation : synthesis of aminooxo-acids through a Mycobacterium smegmatis acyltransferase

A highly-productive strategy based on the use of an acyltransferase from Mycobacterium smegmatis (MsAcT), for the preparation of aminooxo-acids in water was developed. 1 M-scale biotransformations were carried out with excellent yields (68-94%) and rapid reactions (0.5-5 h) starting from anilines and a range of different anhydrides. The high substrate-to-catalyst ratio (M-substrate/M-catalyst: 25 000), enzymatic stability (one month without any activity loss), and excellent protein purification yields (130 mg from 2 g of wet cell paste) made this process a green and cost-efficient approach, which was successfully applied for the preparation of a key intermediate of SAHA synthesis

Boosting the catalytic performance of a marine yeast in a SpinChem® reactor for the synthesis of perillyl alcohol

A sustainable approach for the reduction of perillaldehyde to perillyl alcohol (POH) through alginate immobilized yeast cell beads has been here developed. The process was optimized in small-scale batch reactions and then scaled up in a rotating bed reactor (SpinChem (R)), enhancing productivity while reducing catalyst loading thanks to better mass transfer and catalyst/substrate interaction (i.e., 90% molar conversion, 8 hours). The biocatalyst biomass was also grown on waste material (molasses) and cultivated using seawater to minimize the environmental impact. By harnessing the potential of immobilized yeast cells in a rotating reactor and utilizing eco-friendly resources, this study exemplifies a sustainable biocatalytic approach that can be extended to other natural terpenes. The incorporation of waste materials and seawater into the process showcases the commitment to increase the sustainability of chemical reactions and aligns with the principles of circular economy

Not only vinegar: Acetic Acid Bacteria as versatile biocatalysts for selective oxidation

Acetic acid bacteria (AAB) are aerobic, Gram-negative microorganisms able to oxidise a wide variety of sugars, alcohols, and polyols with high stereo- and regio-specificity, thanks to the presence of dehydrogenases located in their membrane and periplasmic space1. Moreover, several strains of AABs are also able to produce bacterial cellulose (BC), a natural polymer with high crystallinity and purity, which of particular relevance not only for its mechanical properties but also as an innovative support for enzymatic immobilization. Our research group kept an all-round view on the synthetic potentialities of AAB as biocatalysts for oxidations. Acetobacter malorum, isolated from the mother of vinegar, demonstrated good to excellent conversions (60-100%) in the oxidation of different aryl-aliphatic alcohols to their corresponding acids (Figure 1). This result was obtained either using resting cells or immobilised cells into barium alginate beads, thus allowing to maximize the productivity by using continuous-flow reactors. Among the tested compounds, regiospecific desymmetrization of 1,4-benzenedimethanol to 4-(hydroxymethyl) benzoic acid (m.c. 90%) is particularly noteworthy, since the only available procedures show low conversion (40%) and require heavy metals and harsh conditions

Efficient Enzymatic Preparation of Flavor Esters in Water

A straightforward biocatalytic method for the enzymatic preparation of different flavor esters starting from primary alcohols (e.g., isoamyl, n-hexyl, geranyl, cinnamyl, 2-phenethyl, and benzyl alcohols) and naturally available ethyl esters (e.g., formate, acetate, propionate, and butyrate) was developed. The biotransformations are catalyzed by an acyltransferase from Mycobacterium smegmatis (MsAcT) and proceeded with excellent yields (80-97%) and short reaction times (30-120 min), even when high substrate concentrations (up to 0.5 M) were used. This enzymatic strategy represents an efficient alternative to the application of lipases in organic solvents and a significant improvement compared with already known methods in terms of reduced use of organic solvents, paving the way to sustainable and efficient preparation of natural flavoring agents