Secondary metabolites,secretory structures and biological activity of water celery (Apium nodiflorum (L.) Lag.) growing in central Italy

Apium nodiflorum (L.) Lag. (water celery) is an hydrophytic plant forming dense submerged populations occurring along streams and rivers of Europe. In the present work we provided new insights into the phytochemistry and biology of A. nodiflorum. In particular, we studied the chemical profile of essential oil and polar extracts obtained from the flowering aerial parts of water celery growing in central Italy, together with the essential oil biological activities, namely antimicrobial, antioxidant and cytotoxicity on tumour cells. In addition, we correlated the productivity in secondary metabolites to the secreting structures through a detailed micromorphological study. The essential oil was dominated by two main chemotypes, characterized by myristicin and limonene, respectively. The oils showed significant toxicity on tumour cells, as evidenced by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, with IC₅₀ values in the range 3.8–15.9 μg mL^?1, together with inhibitory effects on Candida albicans (inhibition zones of 10–11 mm). HPLC-MS analysis showed the caffeoylquinic acids and quercetin-3-O-glucoside as the most abundant phenolic compounds. Ducts and vittae were the principal secretory structures of vegetative (leaves and stems) and reproductive (fruits) parts, respectively, storing mainly essential oil. Results of this work provide scientific evidences for the possible valorization and exploitation of water celery.     

Maternal genomic variability of the wild boar (Sus scrofa) reveals the uniqueness of East‐Caucasian and Central Italian populations

The phylogeography of the European wild boar was mainly determined by postglacial recolonization patterns from Mediterranean refugia after the last ice age. Here we present the first analysis of SNP polymorphism within the complete mtDNA genome of West Russian (n = 8), European (n = 64), and North African (n = 5) wild boar. Our analyses provided evidence of unique lineages in the East‐Caucasian (Dagestan) region and in Central Italy. A phylogenetic analysis revealed that these lineages are basal to the other European mtDNA sequences. We also show close connection between the Western Siberian and Eastern European populations. Also, the North African samples were clustered with the Iberian population. Phylogenetic trees and migration modeling revealed a high proximity of Dagestan sequences to those of Central Italy and suggested possible gene flow between Western Asia and Southern Europe which was not directly related to Northern and Central European lineages. Our results support the presence of old maternal lineages in two Southern glacial refugia (i.e., Caucasus and the Italian peninsula), as a legacy of an ancient wave of colonization of Southern Europe from an Eastern origin.     

Semi-continuous scale-down models for clone and operating parameter screening in perfusion bioreactors

Perfusion cell culture, confined traditionally to the production of fragile molecules, is currently gaining broader attention in the biomanufacturing of therapeutic proteins. The development of these processes is made difficult by the limited availability of appropriate scale-down models. This is due to the continuous operation that requires complex control and cell retention capacity. For example, the determination of an optimal perfusion and bleed rate for continuous cell culture is often performed in scale-down bioreactors and requires a substantial amount of time and effort. To increase the experimental throughput and decrease the required workload, a semi-continuous procedure, referred to as the VCD_max (viable cell density) approach, has been developed on the basis of shake tubes (ST) and deepwell plates (96-DWP). Its effectiveness has been demonstrated for 12 different CHO-K1-SV cell lines expressing an IgG1. Further, its reliability has been investigated through proper comparisons with perfusion runs in lab-scale bioreactors. It was found that the volumetric productivity and the CSPR_min (cell specific perfusion rate) determined using the ST and 96-DWP models were successfully (mostly within the experimental error) confirmed in lab-scale bioreactors, which then covered a significant scale-up from the half milliliter to the liter scale. These scale-down models are very useful to design and scale-up optimal bioreactor operating conditions as well as screening for different media and cell lines.     

Rational design and characterization of bioplastics from Hermetia illucens prepupae proteins

In this study proteins extracted from prepupae of Hermetia illucens, also known as black soldier fly, are investigated as promising base for a new type of bioplastics for agricultural purposes. Design of experiments techniques are employed to perform a rational study on the effects of different combination of glycerol as plasticizer, citric acid as cross-linking agent and distilled water as solvent on the capability of proteins to form a free-standing film through casting technique, keeping as fixed the quantity of proteins. Glycerol shows interesting properties as plasticizer contributing to the formation of homogenous and free-standing film. Moreover, mechanical and thermal characterizations are performed to estimate the effect of increasing amounts of proteins on the final properties and thickness of the specimens. Proteins derived from H. illucens can be successfully employed as base for bioplastics to be employed for agricultural purposes.     

A new generation of predictive models: The added value of hybrid models for manufacturing processes of therapeutic proteins

Due to the lack of complete understanding of metabolic networks and reaction pathways, establishing a universal mechanistic model for mammalian cell culture processes remains a challenge. Contrarily, data-driven approaches for modeling these processes lack extrapolation capabilities. Hybrid modeling is a technique that exploits the synergy between the two modeling methods. Although mammalian cell cultures are among the most relevant processes in biotechnology and indeed looks ideal for hybrid modeling, their application has only been proposed but never developed in the literature. This study provides a quantitative assessment of the improvement brought by hybrid models with respect to the state-of-the-art statistical predictive models in the context of therapeutic protein production. This is illustrated using a dataset obtained from a 3.5 L fed-batch experiment. With the goal to robustly define the process design space, hybrid models reveal a superior capability to predict the time evolution of different process variables using only the initial and process conditions in comparison to the statistical models. Hybrid models not only feature more accurate prediction results but also demonstrate better robustness and extrapolation capabilities. For the future application, this study highlights the added value of hybrid modeling for model-based process optimization and design of experiments.     

Grandisol as an attractant for the sugar beet pest Bothynoderes affinis and data on other Lixinae species

As a result of field tests in Bulgaria and Hungary, cis‐2‐isopropenyl‐l‐methylcyclobutane ethanol (racemic grandisol) is reported for the first time as an attractant for Bothynoderes affinis (Schrank) (Coleoptera: Curculionidae, Lixinae), a member of the pest weevil complex of sugar beet. Dose–response experiments in the field using Csalomon TAL (modified pitfall) traps (Plant Protection Institute, CAR HAS, Budapest, Hungary) showed that catches of B. affinis adults increased with increasing attractant dose. In a subsequent experiment studying the effect of trap color (white, blue, yellow, fluorescent yellow, and transparent) all traps with the lure caught more than non‐baited control traps, and the highest number of adults was recorded in transparent and yellow baited traps. Trap color had a significant effect on the number of B. affinis females captured. Transparent TAL traps baited with 1–10 mg grandisol applied on rubber dispensers are recommended for the detection and monitoring of B. affinis. In addition to the target species, 17 other Lixinae species were captured during the field experiments, demonstrating for the first time the possible role of grandisol in the chemical communication systems of some of these species. A second locality of Lixus punctiventris Boheman (Lixinae, Lixini) in Bulgaria is reported. TAL traps baited with grandisol might be a useful tool for surveying Lixinae diversity in different biotopes.     

Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase

Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the ‘DFG-out’ inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the ‘gatekeeper’ V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET.