Nonribosomal peptide synthetases (NRPSs) are giant enzymatic assembly lines that deliver many pharmaceutically valuable natural products, including antibiotics. As the search for new antibiotics motivates attempts to redesign nonribosomal metabolic pathways, more robust and rapid sorting and screening platforms are needed. Here, we establish a microfluidic platform that reliably detects production of the model nonribosomal peptide gramicidin S. The detection is based on calcein-filled sensor liposomes yielding increased fluorescence upon permeabilization. From a library of NRPS mutants, the sorting platform enriches the gramicidin S producer 14.5-fold, decreases internal stop codons 250-fold, and generates enrichment factors correlating with enzyme activity. Screening for NRPS activity with a reliable non-binary sensor will enable more sophisticated structure-activity studies and new engineering applications in the future.

During placental formation, cytotrophoblasts (CTBs) fuse into multinucleate, microvilli-coated syncytiotrophoblasts (STBs), which contact maternal blood, mediating nutrient, metabolite, and gas exchange between mother and fetus, and providing a barrier against fetal infection. Trophoblasts remodel the surrounding extracellular matrix through the secretion of matrix metalloproteinases (MMPs). Maternal obesity and diabetes mellitus can negatively impact fetal development and may impair trophoblast function. We sought to model the impact of metabolic stress on STB function by examining MMP and hormone secretion. The BeWo CTB cell line was syncytialized to STB-like cells with forskolin. Cell morphology was examined by electron microscopy and immunofluorescence; phenotype was further assessed by ELISA and RT-qPCR. STBs were exposed to a metabolic stress cocktail (MetaC: 30 mM glucose, 10 nM insulin, and 0.1 mM palmitic acid). BeWo syncytialization was demonstrated by increased secretion of HCGβ and progesterone, elevated syncytin gene expression (ERVW-1 and ERVFRD-1), loss of tight junctions, and increased surface microvilli. MetaC strongly suppressed syncytin gene expression (ERVW-1 and ERVFRD-1), suppressed HCGβ and progesterone secretion, and altered both MMP-9 and MMP-2 production. Metabolic stress modeling diabetes and obesity altered BeWo STB hormone and MMP production in vitro.

Biocatalytic processes are highly selective and specific. However, their utility is limited by the comparatively narrow scope of enzyme-catalysed transformations. To expand product scope, we are developing biocompatible processes that combine biocatalytic reactions with chemo-catalysis in single-flask processes. Here, we show that a chemocatalysed Pictet-Spengler annulation can be interfaced with biocatalysed alcohol oxidation. This two-step, one-pot cascade reaction converts tyramine and aliphatic alcohols to tetrahydroisoquinoline alkaloids in aqueous buffer at mild pH. Tryptamine derivatives are also efficiently converted to tryptolines. Optimization of stoichiometry, pH, reaction time, and whole-cell catalyst deliver the tetrahydroisoquinolines and tryptolines in >90% and >40% isolated yield, respectively, with excellent regioselectivity.

Insulin has been at the forefront of scientific breakthroughs in the past century. In this article, we highlight the pivotal role of chemical biology in driving these innovations and discuss how it continues to shape the future trajectory of insulin research.

Abstract

Since its discovery in 1921, insulin has been at the forefront of scientific breakthroughs. From its amino acid sequencing to the revelation of its three-dimensional structure, the progress in insulin research has spurred significant therapeutic breakthroughs. In recent years, protein engineering has introduced innovative chemical and enzymatic methods for insulin modification, fostering the development of therapeutics with tailored pharmacological profiles. Alongside these advances, the quest for self-regulated, glucose-responsive insulin remains a holy grail in the field. In this article, we highlight the pivotal role of chemical biology in driving these innovations and discuss how it continues to shape the future trajectory of insulin research.

The bioorthogonal toolbox comprises reaction handles for click reactions (CuAAC), strain-promoted reactions (IEDDA cycloaddition), and enzymatic reactions (Staudinger ligation). These reactions have revolutionized the field of chemical biology by providing researchers with powerful tools to investigate and manipulate biomolecules within living systems with precision and control.

Abstract

Bioorthogonal chemistry is a rapidly expanding field of research that involves the use of small molecules that can react selectively with biomolecules in living cells and organisms, without causing any harm or interference with native biochemical processes. It has made significant contributions to the field of biology and medicine by enabling selective labeling, imaging, drug targeting, and manipulation of bio-macromolecules in living systems. This approach offers numerous advantages over traditional chemistry-based methods, including high specificity, compatibility with biological systems, and minimal interference with biological processes. In this review, we provide an overview of the recent advancements in bioorthogonal chemistry and their current and potential applications in translational research. We present an update on this innovative chemical approach that has been utilized in cells and living systems during the last five years for biomedical applications. We also highlight the nucleic acid-templated synthesis of small molecules by using bioorthogonal chemistry. Overall, bioorthogonal chemistry provides a powerful toolset for studying and manipulating complex biological systems, and holds great potential for advancing translational research.

The chemical 5-hydroxymethyl­furfural (HMF) can be derived from lignocellulose and is an interesting bio-based platform chemical as it has the potential to be transformed into numerous valuable building blocks such as the polymer-precursor 2,5-diformylfuran (DFF). To date, only few oxidases acting on HMF are known and by sampling atypical species, we discovered a novel flavin-dependent oxidoreductase from the honeybee Apis mellifera (beeHMFO). The enzyme can perform the chemo­selective oxidation of HMF to DFF but can also readily accept other aromatic alcohols as substrates. The function of the enzyme may well be the antimicrobial generation of hydrogen peroxide using HMF, which is very abundant in honey. The discovery of this insect-derived flavoprotein oxidase holds promising potential in the synthesis of renewable products and demonstrates that insects can be an interesting source for novel biocatalysts.

Gold nanoparticles (AuNPs) consisting of metallic gold are applied in various fields owing to their characteristic physical properties. Collimonas sp. D-25 (D-25) is a Gram-negative bacterium obtained from soil, compost, and other environmental materials in the Akita Prefecture. DP-1 is a water-soluble protein found in D-25 that binds specifically to AuNPs and retains the nano-sized AuNPs with high stability. This study aimed to identify the part of DP-1 interacting with AuNPs and determine its 3D structure in solution using nuclear magnetic resonance (NMR). The peptide fragments obtained by trypsin digestion were examined for their AuNP-binding capacity to determine the key Au-binding domain of DP-1. A fragment consisting of 16 amino acid residues (GHAATPEQYGVVTANK) was identified as the peptide with the highest binding activity. Structural analyses of this peptide indicated that the main chain was elongated, and negatively charged residues in the side chain were exposed on the surface by incorporating AuNPs. These results suggest that DP-1 interacts with AuNPs via negatively charged residues and extended hydrophobic residues for protein-protein interactions. The structural data also provide new insights into biomimetic technologies.

Heavy metal arsenic is a water pollutant that affects millions of lives worldwide. A novel aptamer candidate for specific and sensitive arsenic detection was identified using Graphene Oxide-SELEX (GO-SELEX). Eleven rounds of GO-SELEX were performed to screen As(III) specific sequences. The selected aptamer sequences were evaluated for their binding affinity. The dissociation constant of the best aptamer candidate, As-06[1], was estimated by fluorescence recovery upon target addition, and it was found to be 8.15 nM. A QCM-based biosensing platform was designed based on the target-triggered release of aptamer from the QCM electrode. An rGO-SWCNT nanocomposite was adsorbed on the gold surface, and the single-stranded probe was stacked on the rGO-CNT layer. Upon addition of the target to the solution, a concentration-dependent release of the ssDNA probe was observed and recorded as the change in the electrode frequency. The developed QCM sensor showed a dynamic linear range from 10 nM to 100 nM and a low detection limit of 8.6 nM. The sensor exhibited excellent selectivity when challenged with common interfering anions and cations.       [1] The Provisional IN Patent Application No.: 202311048776, titled An arsenic-binding aptamer and method of preparing the same, filed on July 20, 2023.

Lipopolysaccharides from the commensal gut-associated microbiota are interesting biomolecules for the treatment of various inflammatory diseases. The recent synthesis of lipopolysaccharides from gut-associated lymphoid-tissue-resident Alcaligenes faecalis and Bacteroides vulgatus are highlighted.

Abstract

Lipopolysaccharides from the commensal gut-associated microbiota are interesting biomolecules for the treatment of various inflammatory diseases. Different from pathogenic lipopolysaccharides, commensal lipopolysaccharides have distinct chemical structures and mediate beneficial homeostasis with the immune system of the host. However, the accessibility issues of homogenous and pure commensal lipopolysaccharides hampered the in-depth studies of their functions. In this concept article, we highlight the recent synthesis of lipopolysaccharides from gut-associated lymphoid-tissue-resident Alcaligenes faecalis and Bacteroides vulgatus, which hopes to inspire the more efforts devoting to these fantastic biomolecules.

The quasi-diamond platelet-shaped ZnO (neZnO) nanostructures exhibited enhanced antibacterial activity against Escherichia coli (G−) and Staphylococcus aureus (G+) compared to the quasi-round particles. The morphology of the material plays an important or determining role in the antimicrobial activity, but not the enrichment of oxygen vacancies in neZnO.

Abstract

The current study compares the antibacterial activity of zinc oxide nanostructures (neZnO). For this purpose, two bacterial strains, Escherichia coli (ATCC 4157) and Staphylococcus aureus (ATCC 29213) were challenged in room light conditions with the aforementioned materials. Colloidal and hydrothermal methods were used to obtain the quasi-round and quasi-diamond platelet-shape nanostructures. Thus, the oxygen vacancy (V_O) effects on the surface of neZnO are also considered to assess its effects on antibacterial activity. The neZnO characterization was achieved by X-ray diffraction (XRD), a selected area electron diffraction (SAED) and Raman spectroscopy. The microstructural effects were monitored by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Furthermore, optical absorption ultraviolet visible spectrophotometry (UV-Vis) and X-ray photoelectron spectroscopy (XPS) analyses complement the physical characterization of these nanostructures; neZnO caused 50 % inhibition (IC₅₀) at concentrations from 0.064 to 0.072 mg/mL for S. aureus and from 0.083 to 0.104 mg/mL for E. coli, indicating an increase in activity against S. aureus compared to E. coli. Consequently, quasi-diamond platelet-shaped nanostructures (average particle size of 377.6±10 nm) showed enhanced antibacterial activity compared to quasi-round agglomerated particles (average size of 442.8±12 nm), regardless of Vo presence or absence.