Cell-Free Exploration of the Natural Product Chemical Space

Natural products and secondary metabolites comprise an indispensable resource from living organisms that have transformed areas of medicine, agriculture, and biotechnology. Recent advances in high-throughput DNA sequencing and computational analysis suggest that the vast majority of natural products remain undiscovered. To accelerate the natural product discovery pipeline, cell-free metabolic engineering approaches used to develop robust catalytic networks are being repurposed to access new chemical scaffolds, and new enzymes capable of performing diverse chemistries. Such enzymes could serve as flexible biocatalytic tools to further expand the unique chemical space of natural products and secondary metabolites, and provide a more sustainable route to manufacture these molecules. Herein, we highlight select examples of natural product biosynthesis using cell-free systems and propose how cell-free technologies could facilitate our ability to access and modify these structures to transform synthetic and chemical biology.     

Cellular Assays with a Molecular Endpoint Measured by SAMDI Mass Spectrometry

Cell‐based, high‐throughput screening (HTS) assays are increasingly important tools used in drug discovery, but frequently rely on readouts of gene expression or phenotypic changes and require development of specialized, labeled reporters. Here a cell‐based, label‐free assay compatible with HTS is introduced that can report quantitatively on enzyme activities by measuring mass changes of substrates with matrix‐assisted laser desorption/ionization mass spectrometry. The assay uses self‐assembled monolayers to culture cells on arrays presenting substrates, which serve as reporters for a desired enzyme activity. Each spot of cells is treated with a compound, cultured and lysed, enabling endogenous enzymes to act on the immobilized peptide substrate. It is demonstrated that the assay can measure protein tyrosine phosphatase (PTP) activity from as few as five cells and a screen is described that identifies a compound that reduces PTP activity in cell lysates. This approach offers a valuable addition to the methods available for cell‐based screening.     

A Modelling Approach to Forecast the Effect of Climate Change on the Tagus-Segura Interbasin Water Transfer

This study was conducted in the upper Tagus River basin (UTRB), whose available water resources are partially transferred from the Entrepeñas and Buendía reservoirs after local needs satisfaction to the Segura River basin using the Tagus-Segura water transfer (TSWT), the largest hydraulic infrastructure in Spain. This study evaluates the climate change impact on the TSWT by considering future evaporation rates and bathymetric changes in the Entrepeñas and Buendía reservoirs. The findings of this study indicate a consistent decline in precipitation and an increase in temperature and evaporation under all climate impact scenarios. Consequently, inflows to the reservoirs will decline by 19% (RCP 4.5) and 53% (RCP 8.5) for 2070–2099, which could reduce water volumes that could be transferred to the Segura basin by more than 60%. The simulation of the TSWT operation rules, taking into account the impact of future evaporation and bathymetric changes, demonstrates an additional increase in reductions of water transfer of around 4%, which reveals the need to consider these effects in hydrological planning.

     

Aging process of polyamidoamine dendrimers: Effect of pH and shaking in the fluorescence emission and aggregation-state

In the last years, it has been discovered and intensely studied the non-traditional intrinsic luminescence of polyamidoamine (PAMAM) dendrimers. Nevertheless, their aging process in aqueous suspension is scarcely studied, being unknown the causes of the changes in their luminescence properties. Hence, this work aims to characterize the amine-terminated (DG4.0) and carboxylic acid-terminated (DG4.5) PAMAM dendrimers of generations 4.0 and 4.5, respectively, through the aging process at three different pH conditions, stored with or without shaking. The UV–Vis absorption, the fluorescence emission, and the dendrimer-size distribution are studied for up to 16 days. In a different way than the already published works, this work demonstrates that there is no chemical change in dendrimers through the aging process, even though changes in fluorescence emission are observed. Besides, the changes in the agglomeration patterns of dendrimers are not related to the change in the fluorescence emission through aging. Moreover, large aggregates of DG4.5 are present in water and need to be disrupted by shaking before an in vivo administration.     

Sperm Methylome Profiling Can Discern Fertility Levels in the Porcine Biomedical Model

A combined Genotyping By Sequencing (GBS) and methylated DNA immunoprecipitation (MeDIP) protocol was used to identify—in parallel—genetic variation (Genomic-Wide Association Studies (GWAS) and epigenetic differences of Differentially Methylated Regions (DMR) in the genome of spermatozoa from the porcine animal model. Breeding boars with good semen quality (n = 11) and specific and well-documented differences in fertility (farrowing rate, FR) and prolificacy (litter size, LS) (n = 7) in artificial insemination programs, using combined FR and LS, were categorized as High Fertile (HF, n = 4) or Low Fertile (LF, n = 3), and boars with Unknown Fertility (UF, n = 4) were tested for eventual epigenetical similarity with those fertility-proven. We identified 165,944 Single Nucleotide Polymorphisms (SNPs) that explained 14–15% of variance among selection lines. Between HF and LF individuals (n = 7, 4 HF and 3 LF), we identified 169 SNPs with p ≤ 0.00015, which explained 58% of the variance. For the epigenetic analyses, we considered fertility and period of ejaculate collection (late-summer and mid-autumn). Approximately three times more DMRs were observed in HF than in LF boars across these periods. Interestingly, UF boars were clearly clustered with one of the other HF or LF groups. The highest differences in DMRs between HF and LF experimental groups across the pig genome were located in the chr 3, 9, 13, and 16, with most DMRs being hypermethylated in LF boars. In both HF and LF boars, DMRs were mostly hypermethylated in late-summer compared to mid-autumn. Three overlaps were detected between SNPs (p ≤ 0.0005, n = 1318) and CpG sites within DMRs. In conclusion, fertility levels in breeding males including FR and LS can be discerned using methylome analyses. The findings in this biomedical animal model ought to be applied besides sire selection for andrological diagnosis of idiopathic sub/infertility.     

Effective Separation of CO2 Using Metal-Incorporated rGO Membranes

Graphene-based materials, primarily graphene oxide (GO), have shown excellent separation and purification characteristics. Precise molecular sieving is potentially possible using graphene oxide-based membranes, if the porosity can be matched with the kinetic diameters of the gas molecules, which is possible via the tuning of graphene oxide interlayer spacing to take advantage of gas species interactions with graphene oxide channels. Here, highly effective separation of gases from their mixtures by using uniquely tailored porosity in mildly reduced graphene oxide (rGO) based membranes is reported. The gas permeation experiments, adsorption measurement, and density functional theory calculations show that this membrane preparation method allows tuning the selectivity for targeted molecules via the intercalation of specific transition metal ions. In particular, rGO membranes intercalated with Fe ions that offer ordered porosity, show excellent reproducible N₂/CO₂ selectivity of ≈97 at 110 mbar, which is an unprecedented value for graphene-based membranes. By exploring the impact of Fe intercalated rGO membranes, it is revealed that the increasing transmembrane pressure leads to a transition of N₂ diffusion mode from Maxwell–Stefan type to Knudsen type. This study will lead to new avenues for the applications of graphene for efficiently separating CO₂ from N₂ and other gases.     

Sunflower Lecithin: Application of a Fractionation Process with Absolute Ethanol

Native or modified lecithins are widely used as a multifunctional ingredient in the food industry. A fractionation process of sunflower lecithin (a non GMO product) with absolute ethanol was used for obtaining enriched fractions in certain phospholipids under different experimental conditions (temperature 35–65 °C, time of fractionation 30–90 min, ethanol/lecithin ratio 2:1, 3:1). Phospholipid enrichment in PC and PI fractions was obtained and analyzed by ³¹P NMR determinations. The percent extraction coefficients for different phospholipids (%E_PC, %E_PE and %E_PI) in both fractions were calculated. Values of %E_PC in PC fractions significantly increased (p < 0.05) from 12.8 (35 °C, 30 min, 2:1) to 57.7 (65 °C, 90 min, 3:1) at increasing temperature and incubation time. %E_PE varied from 3.0 to 18.3 in the same fraction while %E_PI presented lower values (<3%) under all the conditions assayed. The study of the effect of the operating conditions on the fractionation process evidenced a relevant influence of temperature, incubation time and to a minor extent of the ethanol/lecithin ratio on the enriched fraction yield% and selectivity of the main phospholipids (PC, PI, PE) estimated by %E_PL. Response surface methodology (RSM) was utilized to explain the influence of the different parameters to optimize this process.     

Discovery of a Class of Diketopiperazines as Antiprion Compounds

Prion diseases are fatal neurodegenerative and infectious disorders for which effective pharmacological tools are not yet available. This unmet challenge and the recently proposed interplay between prion diseases and Alzheimer's have led to a more urgent demand for new antiprion agents. Herein, we report the identification of a novel bifunctional diketopiperazine (DKP) derivative 1 d , which exhibits activity in the low micromolar range against prion replication in ScGT1 cells, while showing low cytotoxicity. Supported by properly addressed molecular modeling studies, we hypothesized that a planar conformation is the major determinant for activity in this class of compounds. Moreover, studies aimed at assessing the mechanism‐of‐action at the molecular level showed that 1 d might interact directly with recombinant prion protein (recPrP) to prevent its conversion to the pathogenic misfolded prion protein (PrP^Sc)‐like form. This investigation suggests that DKP based antiprion compounds can serve as a promising lead scaffold in developing new drugs to combat prion diseases.