Golden Standard: a complete standard, portable, and interoperative MoClo tool for model and non-model proteobacteria.

Modular cloning has become a benchmark technology in synthetic biology. However, a notable disparity exists between its remarkable development and the need for standardization to facilitate seamless interoperability among systems. The field is thus impeded by an overwhelming proliferation of organism-specific systems that frequently lack compatibility. To overcome these issues, we present Golden Standard (GS), a Type IIS assembly method underpinned by the Standard European Vector Architecture. GS unlocks modular cloning applications for most bacteria, and delivers combinatorial multi-part assembly to create genetic circuits of up to twenty transcription units (TUs). Reliance on MoClo syntax renders GS fully compatible with many existing tools and it sets the path towards efficient reusability of available part libraries and assembled TUs. GS was validated in terms of DNA assembly, portability, interoperability and phenotype engineering in α-, ß-, γ- and δ-proteobacteria. Furthermore, we provide a computational pipeline for parts characterization that was used to assess the performance of GS parts. To promote community-driven development of GS, we provide a dedicated web-portal including a repository of parts, vectors, and Wizard and Setup tools that guide users in designing constructs. Overall, GS establishes an open, standardized framework propelling the progress of synthetic biology as a whole.

New Phage-Derived Antibacterial Enzyme PolaR Targeting Rothia spp.

Rothia is an opportunistic pathogen, particularly life-threatening for the immunocompromised. It is associated with pneumonia, endocarditis, peritonitis and many other serious infections, including septicemia. Of note, Rothia mucilaginousa produces metabolites that support and increase overgrowth of Pseudomonas aeruginosa, one of the ESKAPE bacteria. Endolysins are considered as antibacterial enzymes derived from bacteriophages that selectively and efficiently kill susceptible bacteria without harming human cells or the normal microbiome. Here, we applied a computational analysis of metagenomic sequencing data of the gastric mucosa phageome extracted from human patients' stomach biopsies. A selected candidate anti-Rothia sequence was produced in an expression system, purified and confirmed as a Rothia mucilaginosa- and Rothia dentocariosa-specific endolysin PolaR, able to destroy bacterial cells even when aggregated, as in a biofilm. PolaR had no cytotoxic or antiproliferative effects on mammalian cells. PolaR is the first described endolysin selectively targeting Rothia species, with a high potential to combat infections caused by Rothia mucilaginosa and Rothia dentocariosa, and possibly other bacterial groups. PolaR is the first antibacterial enzyme selected from the gastric mucosa phageome, which underlines the biological complexity and probably underestimated biological role of the phageome in the human gastric mucosa.

Fruits of Hippophaë rhamnoides in human leukocytes and Caco-2 cell monolayer models-A question about their preventive role in lipopolysaccharide leakage and cytokine secretion in endotoxemia.

Preparations from Hippophaë rhamnoides L. (sea buckthorn) have been traditionally used in the treatment of skin and digestive disorders, such as gastritis, gastric and duodenal ulcers, uterine erosions, as well as oral, rectal, and vaginal mucositis, in particular in the Himalayan and Eurasian regions. An influence of an aqueous extract from the fruits of H. rhamnoides (HR) on leakage of lipopolysaccharide (LPS) from Escherichia coli through gut epithelium developed from the human colorectal adenocarcinoma (Caco-2) monolayer in vitro and glucose transporter 2 (GLUT2) translocation were the principal objectives of the study. Additionally, the effect of HR on the production of pro- and anti-inflammatory cytokines (interleukins: IL-8, IL-1ß, IL-10, IL-6; tumor necrosis factor: TNF-α) by the Caco-2 cell line, human neutrophils (PMN), and peripheral blood mononuclear cells (PBMC) was evaluated. The concentration of LPS on the apical and basolateral sides of the Caco-2 monolayer was evaluated with a Limulus Amebocyte Lysate (LAL) assay. GLUT2 translocation was evaluated using an immunostaining assay, whereas secretion of cytokines by cell cultures was established with an enzyme-linked immunosorbent (ELISA) assay. HR (500 µg/ml) significantly inhibited LPS leakage through epithelial monolayer in vitro in comparison with non-treated control. The treatment of Caco-2 cells with HR (50-100 µg/ml) showed GLUT2 expression similar to the non-treated control. HR decreased the secretion of most pro-inflammatory cytokines in all tested models. HR might prevent low-grade chronic inflammation caused by metabolic endotoxemia through the prevention of the absorption of LPS and decrease of chemotactic factors released by immune and epithelial cells, which support its use in metabolic disorders in traditional medicine.

Physicochemical Properties and Digestion Resistance of Acetylated Starch Obtained from Annealed Starch.

One of the examples of physical starch modifications is the retention of a starch suspension in water having a temperature slightly lower than the pasting temperature (annealing). The aim of this study was to investigate the effect of the annealing process performed at various temperatures as the first stage of starch modification. The annealed starch preparations were then esterified using acetic acid anhydride. Finally, the annealed and acetylated starch preparations were determined for their properties. The annealing of starch before acetylation triggered changes in the properties of the modified preparations. It contributed to a higher degree of starch substitution with acetic acid residues and to the increased swelling power of starch. Both these properties were also affected by the annealing temperature. The highest resistance to amylolysis was found in the case of the starch preparation annealed at 53.5 °C and acetylated. The double modification involving annealing and acetylation processes increased the onset and end pasting temperatures compared to the acetylation alone. Similar observations were made for the consistency coefficient and yield point.

The Annealing of Acetylated Potato Starch with Various Substitution Degrees.

This study aimed to determine the effect of "annealing" acetylated potato starch with a homogenous granule size and various degrees of substitution on the thermal pasting characteristics (DSC), resistance to amylases, rheology of the prepared pastes, swelling power and dynamics of drug release. A fraction of large granules was separated from native starch with the sedimentation method and acetylated with various doses of acetic anhydride (6.5, 13.0 or 26.0 26 cm3/100 g starch). The starch acetates were then annealed at slightly lower temperatures than their pasting temperatures. The annealing process caused an almost twofold increase in the resistance to amylolysis and a threefold increase in the swelling power of the modified starch preparations. The heat of phase transition decreased almost two times and the range of starch pasting temperatures over two times, but the pasting temperature itself increased by ca. 10 °C. The 40 g/100 g addition of the modified starch preparation decreased the rate of drug release from a hydrogel by ca. one-fourth compared to the control sample.

Effect of the Botanical Origin on Properties of RS3/4 Type Resistant Starch.

This study aimed to compare properties of retrograded starch acetates with an identical degree of substitution, but produced from raw materials of various botanical origin. Retrograded starch was produced from potato, wheat, corn, and tapioca starch, and afterwards acetylated with an acetic acid anhydride, adjusting reagent doses to achieve an identical degree of esterification of the modified preparation (2.1 g/100 g). Preparations of retrograded starch and acetylated retrograded starch differed significantly in their properties, which was due to the disparate botanical origin of starch. The highest susceptibility to acetylation was demonstrated for potato starch, and the lowest one for wheat starch. Acetylation of retrograded starch of various botanical origin increased its solubility in water, swelling power and viscosity of its pastes, as well as decreased its amylose content. Preparations of acetylated retrograded starches of disparate botanical origins may be deemed preparations of RS3/4 type resistant starch because they exhibit significant (23.5⁻34.0%) resistance to the activity of amylolytic enzymes.