Successful stem cell applications could have a significant impact on the medical field, where many lives are at stake. However, the translation of stem cells to the clinic could be improved by overcoming challenges in stem cell transplantation and in vivo retention at the site of tissue damage. This review aims to showcase the most recent insights into developing hydrogels that can deliver, retain, and accommodate stem cells for tissue repair. Hydrogels can be used for tissue engineering, as their flexibility and water content makes them excellent substitutes for the native extracellular matrix. Moreover, the mechanical properties of hydrogels are highly tuneable, and recognition moieties to control cell behaviour and fate can quickly be introduced. This review covers the parameters necessary for the physicochemical design of adaptable hydrogels, the variety of (bio)materials that can be used in such hydrogels, their application in stem cell delivery and some recently developed chemistries for reversible crosslinking. Implementing physical and dynamic covalent chemistry has resulted in adaptable hydrogels that can mimic the dynamic nature of the extracellular matrix.

Non-enzymatic post-translational modifications (nPTMs) have been proposed as indicators of cellular stresses and diseases. Unfortunately, direct assessment of nPTMs in native environment is extremely challenging due to the heterogeneity of adducts and the lack of tagging tools. Given these challenges, bioorthogonal probes (BPs) have been developed for the analysis of nPTMs. The rationality is that BPs could selectively install azides or alkynes into nPTMs as a biorthogonal handle for the following enrichment or tracking. Herein, we review the state-of-art of BPs used for nPTMs studies, clarify their working principles, and highlight how they advance our understanding of the biological functions of nPTMs.

Bioinformatics has become an indispensable tool for natural products research in the genomic era. One of the key challenges is to accurately convert sequence data of a biosynthetic gene cluster into chemical information such as the enzymatic function or the biosynthetic product structure. Type II polyketide synthase is the most bioinformatically well-studied class of non-modular biosynthetic machinery and represents a model system to showcase bioinformatic applications in natural products research. This review takes a bioinformatics-centered perspective and summarizes the past advances and future opportunities of bioinformatics-guided research on type II polyketide synthases. How bioinformatics has contributed to deepen the chemical understanding of type II PKSs will be discussed with the focus on enzymology, evolution, structural prediction of the biosynthetic products, genome mining, and the global analyses of their polyketide products.

Glycosylation in live cell and animal modulate a constellation of biological functions. The advent of Chemical Biology has revolutionized the analysis and tailoring of glycans, by introducing myriads of glycan engineering methods. However, the ideal scenario to achieve glycan monitoring and structural manipulation at any hierarchical levels is unmet yet. Herein we review recent advances in the methodological innovation and the versatile applications of the protein-specific glycan visualization and editing in deciphering the biological functions of glycans. An outlook for future directions toward specific sugar-chain editing is also included.

Liver fibrosis is a process of excessive accumulation of extracellular matrix caused by liver injury. Liver fibrosis can progress to cirrhosis or even liver cancer without proper intervention. Until now, no effective therapeutic drugs have been clinically approved for treating liver fibrosis. Hence, the development of safe and effective antifibrotic drugs is particularly important. As a representative biomaterial, peptides have been investigated as key components for constructing antifibrotic nanomaterials given their advantages of biological origination, synthetic availability, and good biocompatibility. Peptides serve as multifunctional motifs in antifibrotic nanomaterials, such as liver-targeting molecules, antifibrotic molecules, and self-assembling building blocks for the formation of the nanomaterials. In this review, we focus on peptide-based nanoarchitectonics for treating liver fibrosis, including nanomaterials modified with liver-targeting peptides, nanomaterials for the efficient delivery of antifibrotic peptides, and self-assembled peptide nanomaterials for the delivery of antifibrotic drugs. The design rules of these peptide-based nanomaterials are described. The antifibrotic mechanisms and effects of these peptide-based nanomaterials in treating liver fibrosis and related diseases are highlighted. The challenges and future perspectives of using peptide-based nanoarchitectonics for the treatment of liver fibrosis are discussed. These results are expected to accelerate the rational design and clinical translation of antifibrotic nanomaterials.

Whereas directed evolution and rational design by structural inspection are established tools for enzyme redesign, computational methods are less mature but have the potential to predict small sets of mutants with desired properties without laboratory screening of large libraries. We have explored the use of computational enzyme redesign to change the enantioselectivity of a highly thermostable alcohol dehydrogenase from Thermus thermophilus in the asymmetric reduction of ketones. The enzyme reduces acetophenone to (S)-1-phenylethanol. To invert the enantioselectivity, we used an adapted CASCO workflow which included Rosetta for enzyme design and molecular dynamics simulations for ranking. To correct for unrealistic binding modes, we used Boltzmann weighing of binding energies computed by a linear interaction energy approach. This computationally cheap method predicted four variants with inverted enantioselectivity, each with 6-8 mutations around the substrate-binding site, causing only modest reduction (2- to 7-fold) of kcat /KM values. Laboratory testing showed that three variants indeed had inverted enantioselectivity, producing (R)-alcohols with up to 99 % enantiomeric excess. The broad substrate range allowed reduction of acetophenone derivatives with full conversion to highly enantioenriched alcohols. The results demonstrate the use of computational methods to control ketoreductase stereoselectivity in asymmetric transformations with minimal experimental screening.

A major limitation for the development of more effective oligonucleotide therapeutics has been a lack of understanding of their penetration into the cytosol. While prior work has shown how backbone modifications affect cytosolic penetration, it is unclear how cytosolic penetration is affected by other features including base composition, base sequence, length, and degree of secondary structure. We have applied the chloroalkane penetration assay, which exclusively reports on material that reaches the cytosol, to investigate the effects of these characteristics on the cytosolic uptake of druglike oligonucleotides. We found that base composition and base sequence had moderate effects, while length did not correlate directly with the degree of cytosolic penetration. Investigating further, we found that the degree of secondary structure had the largest and most predictable correlations with cytosolic penetration. These methods and observations add a layer of design for maximizing the efficacy of new oligonucleotide therapeutics.

Hofmeister effects play a critical role in numerous physicochemical and biological phenomena, including the solubility and/or accumulation of proteins, the activities of enzymes, ion transport in biochannels, the structure of lipid bilayers, and the dynamics of vesicle opening and exocytosis. This minireview focuses on how ionic specificity affects the physicochemical properties of biomolecules to regulate cellular exocytosis, vesicular content, and nanovesicle opening. We summarize recent progress in further understanding Hofmeister effects on biomacromolecules and their applications in biological systems. These important steps have increased our understanding of the Hofmeister effects on cellular exocytosis, vesicular content, and nanovesicle opening. Increasing evidence is firmly establishing that the ions along the Hofmeister series play an important role in living organisms that has often been ignored.

Messenger RNA (mRNA) is being used as part of an emerging class of biotherapeutics with great promise for preventing and treating a wide range of diseases, as well as encoding programmable nucleases for genome editing. However, mRNA's low stability and immunogenicity, as well as the impermeability of the cell membrane to mRNA greatly limit mRNA's potential for therapeutic use. Lipid nanoparticles (LNPs) are currently one of the most extensively studied nanocarriers for mRNA delivery and have recently been clinically approved for developing mRNA-based vaccines to prevent COVID-19. In this review, we summarize the latest advances in designing ionizable lipids and formulating LNPs for intracellular and tissue-targeted mRNA delivery. Furthermore, we discuss the progress of intracellular mRNA delivery for spatiotemporally controlled CRISPR/Cas9 genome editing by using LNPs. Finally, we provide a perspective on the future of LNP-based mRNA delivery for CRISPR/Cas9 genome editing and the treatment of genetic disorders.

Protein posttranslational modifications (PTMs) play critical roles in regulating cellular activities. Here we provide a survey of genetic code expansion (GCE) methods that were applied in the co-translational installation and studies of PTMs through noncanonical amino acid (ncAA) mutagenesis. We begin by reviewing types of PTM that have been installed by GCE with a focus on modifications of tyrosine, serine, threonine, lysine, and arginine residues. We also discuss examples of applying these methods in biological studies. Finally, we end the piece with a short discussion on the challenges and the opportunities of the field.

The gel-sol transition of self-assembling peptides is a useful switch for environment-dependent drug release. For their applications, kinetics control of the responses is important for matching the velocity of release to the target biological events. Here we demonstrate the chemical control of redox-triggered gel-sol transition kinetics of self-assembling peptides by altering the amino acid sequence. Amphiphilic peptides were developed in which a methionine residue was located in the middle (JigSAP-IMI) or near the N terminus (JigSAP-MII). Both peptides formed hydrogels under physiological conditions-forming β-sheet-based supramolecular nanofibers. In contrast, the oxidized forms remained in the solution state under identical conditions-adopting α-helix-rich secondary structures. Upon oxidation with H2 O2 , a reactive oxygen species, JigSAP-MII showed a faster gel-to-sol transition and cargo-releasing than JigSAP-IMI, thus indicating that the phase-transition and releasing kinetics of self-assembling peptides can be rationally controlled by the position of the reactive amino acid residue.

Antibody-oligonucleotide conjugates (AOCs) are important tools for drug development and biochemical analysis. However, the structural heterogeneity of AOCs synthesized through conventional coupling methods raises reproducibility and safety concerns in clinical trials. To address these issues, different covalent coupling approaches have been developed to synthesize AOCs with precise site-specificity and degree of conjugation. This Concept article categorizes these approaches as linker-free or linker-mediated and provides details on their chemistry and potential applications. Several factors, including site-specificity, conjugation control, accessibility, stability, and efficiency, are highlighted when evaluating the pros and cons of these approaches. The article also discusses the future of AOCs, including the development of better conjugation approaches to ensure stimuli-responsive release and the application of high-throughput methods to facilitate their development.

The introduction of chemical modifications on the nucleic acid scaffold has allowed for the progress of antisense oligonucleotides (ASOs) in the clinic for the treatment of a variety of disorders. In contribution to the repertoire of gene-silencing nucleic acid modifications, herein we report the synthesis and incorporation of C5-propynyl arabinouridine (araUP ) and arabinocytidine (araCP ) into mixed-base ASOs containing a pyrimidine core. Substitution of the core with araUP and araCP resulted in stabilization of the duplex formed with RNA but not with DNA. Similar results were obtained with ASOs bearing phosphorothioate linkages or methoxyethyl (MOE) wings in a gapmer design. All modified ASOs were compatible with E. coli RNase H mediated degradation of target RNA. Substitution of DNA for araUP and araCP in the central portion of a gapmer with MOE wings demonstrated improved nuclease resistance. These results suggest C5-modified arabinonucleic acids may serve as a potential chemical modification for therapeutic ASOs.

S-Adenosylmethionine (SAM) is an enzyme cofactor involved in methylation, aminopropyl transfer, and radical reactions. This versatility renders SAM-dependent enzymes of great interest in biocatalysis. The usage of SAM analogues adds to this diversity. However, high cost and instability of the cofactor impedes the investigation and usage of these enzymes. While SAM regeneration protocols from the methyltransferase (MT) byproduct S-adenosylhomocysteine are available, aminopropyl transferases and radical SAM enzymes are not covered. Here, we report a set of efficient one-pot systems to supply or regenerate SAM and SAM analogues for all three enzyme classes. The systems' flexibility is showcased by the transfer of an ethyl group with a cobalamin-dependent radical SAM MT using S-adenosylethionine as a cofactor. This shows the potential of SAM (analogue) supply and regeneration for the application of diverse chemistry, as well as for mechanistic studies using cofactor analogues.

Oxygen-directed methylation is a ubiquitous tailoring reaction in natural product pathways catalysed by O-methyltransferases (OMTs). Promiscuous OMT biocatalysts are thus a valuable asset in the toolkit for sustainable synthesis and optimization of known bioactive scaffolds for drug development. Here, we characterized the enzymatic properties and substrate scope of two bacterial OMTs from Desulforomonas acetoxidans and Streptomyces avermitilis and determined their crystal structures. Both OMTs methylated a wide range of catechol-like substrates, including flavonoids, coumarins, hydroxybenzoic acids, and their respective aldehydes, an anthraquinone and an indole. One enzyme also accepted a steroid. The product range included pharmaceutically relevant compounds such as (iso)fraxidin, iso(scopoletin), chrysoeriol, alizarin 1-methyl ether, and 2-methoxyestradiol. Interestingly, certain non-catechol flavonoids and hydroxybenzoic acids were also methylated. This study expands the knowledge on substrate preference and structural diversity of bacterial catechol OMTs and paves the way for their use in (combinatorial) pathway engineering.

Cannabidiol (CBD) is a non-intoxicating cannabinoid from cannabis sativa that has demonstrated efficacious against inflammation, which can be considered as a potential drug for arthritis treatment. However, the poor solubility and low bioavailability limit its clinical application. Here, we report an effective strategy to fabricate Cannabidiol-loaded poly(lactic-co-glycolic acid) copolymer (CBD-PLGA) nanoparticles (NPs), with a spherical morphology and an average diameter of 238 nm. CBD was sustained release from CBD-PLGA-NPs, which improved the bioavailability of CBD. The CBD-PLGA-NPs effectively protect the damage of LPS to cell viability. We observed that CBD-PLGA-NPs significantly suppressed LPS-induced primary rat chondrocyte expression of inflammatory cytokines, including interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α) and matrix metalloproteinase 13 (MMP-13). Remarkably, CBD-PLGA-NPs also showed better therapeutic effects of inhibiting the degradation of the extracellular matrix of chondrocytes than equivalent CBD solution. In general, the fabrication CBD-PLGA-NPs showed good protection of primary chondrocytes in vitro and is a promising system for osteoarthritis treatment.

Chemically labile ester linkages can be introduced into lignin by incorporation of monolignol conjugates, which are synthesized in planta by acyltransferases that use a coenzyme A (CoA) thioester donor and a nucleophilic monolignol alcohol acceptor. The presence of these esters facilitates processing and aids in the valorization of renewable biomass feedstocks. However, the effectiveness of this strategy is potentially limited by the low steady-state levels of aromatic acid thioester donors in plants. As part of an effort to overcome this, aromatic acid CoA ligases involved in microbial aromatic degradation were identified and screened against a broad panel of substituted cinnamic and benzoic acids involved in plant lignification. Functional fingerprinting of this ligase library identified four robust, highly active enzymes capable of facile, rapid, and high-yield synthesis of aromatic acid CoA thioesters under mild aqueous reaction conditions mimicking in planta activity.

One of the main problems in developing immunosensors featuring carbon nanotubes (CNTs) is immobilizing antibodies (Abs) onto the CNT surface to afford selective binding to target antigens (Ags). In this work, we developed a practical supramolecular Ab conjugation strategy based on resorc[4]arene modifiers. To improve the Ab orientation on the CNTs surface and optimizing the Ab/Ag interaction, we exploited the host-guest approach by synthesizing two newly resorc[4]arene linkers R1 and R2 via well-established procedures. The upper rim was decorated with eight methoxyl groups to promote selective recognition of the fragment crystallizable (Fc ) region of the Ab. Moreover, the lower rim was functionalized with 3-bromopropyloxy or 3-azidopropiloxy substituents to bind the macrocycles on the multi-walled carbon nanotubes (MWCNTs) surface. Accordingly, several chemical modifications of MWCNTs were evaluated. After the morphological and electrochemical characterization of nanomaterials, the resorc[4]arene-modified MWCNTs were deposited onto a glassy carbon electrode surface to evaluate their potential applicability for label-free immunosensor development. The most promising system showed an improved electrode active area (AEL ) of almost 20 % and a site-oriented immobilization of the SARS-CoV-2 spike protein S1 antibody (Ab-SPS1). The developed immunosensor revealed a good sensitivity (23.64 μA mL ng-1  cm-2 ) towards the SPS1 antigen and a limit of detection (LOD) of 1.01 ng mL-1 .

The formal asymmetric and stereodivergent enzymatic reduction of α-angelica lactone to both enantiomers of γ-valerolactone was achieved in a one-pot cascade by uniting the promiscuous stereoselective isomerization activity of Old Yellow Enzymes with their native reductase activity. In addition to running the cascade with one enzyme for each catalytic step, a bifunctional isomerase-reductase biocatalyst was designed by fusing two Old Yellow Enzymes, thereby generating an unprecedented case of an artificial enzyme catalyzing the reduction of nonactivated C=C bonds to access (R)-valerolactone in overall 41 % conversion and up to 91 % ee. The enzyme BfOYE4 could be used as single biocatalyst for both steps and delivered (S)-valerolactone in up to 84 % ee and 41 % overall conversion. The reducing equivalents were provided by a nicotinamide recycling system based on formate and formate dehydrogenase, added in a second step. This enzymatic system provides an asymmetric route to valuable chiral building blocks from an abundant bio-based chemical.

A chemical synthesis of two novel phenyl glycosides of trisaccharides related to acetylarabinoxylan is described. The trisaccharides bear acetyl and arabinofuranosyl moieties at the non-reducing-end xylopyranosyl residue, which is substituted at positions 2 and 3. Both compounds were treated with various xylan deacetylases classified in different carbohydrate esterase (CE) families and significant differences between the families were found. While the arabinosylation hampers deacetylation by CE2-CE5 and CE12 family members, both epitopes are deesterified by CE1 and in particular CE6 enzymes. The 3-O-acetylated 2-O-arabinosylated compound is also processed by CE7 and majority of CE16 esterases, but not by a hitherto non-classified Flavobacterium johnsoniae acetylxylan esterase. The data suggests that a slow deesterification of the 2-O-acetylated 3-O-arabinosylated compound may be due to the acetyl group migration followed by deacetylation of this migration product.

Here we describe a novel catalyst-free 1,3-dipolar cycloaddition bioconjugation approach for chemical modification of proteins. The dehydroalanine (Dha)-containing protein reacts with nitrile oxides generated in situ through 1,3-dipolar cycloaddition in fully aqueous-buffered systems. This leads to the formation of a new isoxazoline ring at a pre-defined site (Dha) of the protein. Furthermore, the 1-pyrene isoxazoline-installed annexin V acts as a fluorescent probe, which successfully labels the outer cellular membranes of human cholangiocarcinoma (HuCCA-1) cells for detection of apoptosis.

Chemical biology is a steadily growing field that has traditionally struggled to clearly define its boundaries in a short sentence. However, it can be stated that through the development of chemical and physicochemical tools, concepts and methods, chemical biology aims to address or stimulate biological questions at the molecular level in living organisms. Chemical biologists design and develop molecular tools that can probe or modulate biological processes, in order to understand their function, and sometimes to modify it for specific applications, but also to observe and analyze these tools in complex biological environments. Essentially positioned as a fundamental approach, chemical biology often remains very close to potential applications as it builds molecular objects capable of reacting to a significant biological stimulus. Chemical biology therefore finds natural development in fields such as health for the design of drugs and diagnostic systems or the environment for applications in crop science and ecology.

How should lab heads train their students and what is the role of degree-granting institutions in this process? Chua, Vandana, and Hsieh discuss the needs of graduate students and ways to cultivate purposeful, personalized mentee training in the lab.

Examination of the genetic code (GeCo) reveals that amino acids coded by (A/U) codons display a large functional spectrum and bind RNA whereas, except for Arg, those coded by (G/C) codons do not. From a stereochemical viewpoint, the clear preference for (A/U)-rich codons to be located at the GeCo half blocks suggests they were specifically determined. Conversely, the overall lower affinity of cognate amino acids for their (G/C)-rich anticodons points to their late arrival to the GeCo. It is proposed that i) initially the code was composed of the eight (A/U) codons; ii) these codons were duplicated when G/C nucleotides were added to their wobble positions, and three new codons with G/C in their first position were incorporated; and iii) a combination of A/U and G/C nucleotides progressively generated the remaining codons.

Phase separation is a crucial biophysical process that governs cellular signaling and function. This process allows biomolecules to separate and form membraneless compartments in response to both extra- and intra-cellular stimuli. Recently, the identification of phase separation in different immune signaling pathways, including the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway, has shed light on its tight association with pathological processes such as viral infections, cancers, and inflammatory diseases. In this review, we present the phase separation in cGAS-STING signaling, along with its related cellular regulatory functions. Furthermore, we discuss the introduction of therapeutics targeting cGAS-STING signaling, which plays a pivotal role in cancer progression.

RNA function is increasingly appreciated to be more complex than merely communicating between DNA sequence and protein structure. RNA localization has emerged as a key contributor to the intricate roles RNA plays in the cell, and the link between dysregulated spatiotemporal localization and disease warrants an exploration beyond sequence and structure. However, the tools needed to visualize RNA with precise resolution are lacking in comparison to methods available for studying proteins. In the past decade, many techniques have been developed for imaging RNA, and in parallel super resolution and single-molecule techniques have enabled imaging of single molecules in cells. Of these methods, single molecule localization microscopy (SMLM) has shown significant promise for probing RNA localization. In this review, we highlight current approaches that allow super resolution imaging of specific RNA transcripts and summarize challenges and future opportunities for developing innovative RNA labeling methods that leverage the power of SMLM.

Ring-closure is a key step in current pyrimidine anabolism and one may wonder whether cyclisation reactions could be promoted in the geochemical context at the origins of life, i. e. with the help of minerals. Various prebiotic minerals were tested in this work, including silica, carbonates, microporous minerals. In particular, the role of zinc ions supported on minerals was investigated in view of its presence in the catalytic site of cyclic amidohydrolase enzymes. Based on in situ (TGA: ThermoGravimetric Analysis, ATR-IR: Attenuated Total Reflectance-InfraRed) and ex situ (1 H NMR- Nuclear Magnetic Resonance) characterisations, we identified the products of thermal activation of NCA (N-carbamoyl-aspartic acid) in wetting-and-drying scenarios on the surface of minerals. NCA can cyclize extensively only on some surfaces, with the predominant product being 5-carboxymethylhydantoin (Hy) rather than dihydroorotate (DHO), while there is a competition with hydrolysis on others. Replacing the enzymes with heterogeneous catalysts also works with other reactions catalysed by enzymes of the cyclic amidohydrolases family. The role of the hydrophilicity/hydrophobicity of minerals as well as the regioselectivity of the cyclisation (5-carboxymethylhydantoin versus dihydroorotate) are examined.

Covalent catalytic intermediates provide valuable information for revealing the catalytic mechanism, probing the enzyme activity and identifying substrate specificity. However, naturally formed covalent intermediates are too rapidly degraded for general biological studies. In order to capture transient covalent intermediates, a variety of chemical strategies have been developed over decades to extend the half-life of the enzyme-substrate intermediates (or close analogues) required for the downstream structural and functional studies. This review summarizes three general mechanism-based strategies for trapping covalent catalytic intermediates. In particular, enzyme mutant-based approaches, especially the introduction of genetically encoded 2,3-diaminopropionic acid to replace the catalytic cysteine/serine in proteases for acyl-enzyme intermediate trapping are described. In addition, the applications of trapped intermediates in structural, functional and protein labeling studies are presented, and the potential new directions of using enzyme substrate traps are discussed at the end of the review.

Mycosporine-like amino acids (MAAs) are natural UV-absorbing sunscreens that evolved in cyanobacteria and algae to palliate harmful effects from obligatory exposure to solar radiation. Multiple lines of evidence prove that in cyanobacteria all MAAs are derived from mycosporine-glycine, which is typically modified by an ATP-dependent ligase encoded by the gene mysD. The function of the mysD ligase has been experimentally described but haphazardly named based solely upon sequence similarity to the d-alanine-d-alanine ligase of bacterial peptidoglycan biosynthesis. Combining phylogeny and alpha-fold tertiary protein structure prediction unambiguously distinguished mysD from d-alanine-d-alanine ligase. The renaming of mysD to mycosporine-glycine-amine ligase (MG-amine ligase) using recognised enzymology rules of nomenclature is, therefore, proposed, and considers relaxed specificity for several different amino acid substrates. The evolutionary and ecological context of MG-amine ligase catalysis merits wider appreciation especially when considering exploiting cyanobacteria for biotechnology, for example, producing mixtures of MAAs with enhanced optical or antioxidant properties.

Cancer vaccines are based on a vaccinology strategy whereby the patient's immune system is harnessed to induce a specific immune response to kill cancer cells and comprises two categories: prophylactic and therapeutic. Glycoprotein mucin 1 (MUC1), which is overexpressed and poorly glycosylated on cancer cells, is one of the most promising candidates for the development of new cancer vaccines. However, it should be noted that mucin-like glycopeptides are poorly immunogenic and unable to elicit effective and long-lasting immune responses. Therefore, MUC1-derived tumor antigens need to be conjugated with immune activators. This review focuses on the synthesis of MUC1 glycopeptides, provides an overview of recently advanced designs of vaccines based on MUC1, and compares the advantages and disadvantages of the various strategies devised to date.

Degraders have illustrated that compound-induced proximity to E3 ubiquitin ligases can prompt the ubiquitination and degradation of disease-relevant proteins. Hence, this pharmacology is becoming a promising alternative and complement to available therapeutic interventions (e. g., inhibitors). Degraders rely on protein binding instead of inhibition and, hence, they hold the promise to broaden the druggable proteome. Biophysical and structural biology approaches have been the cornerstone of understanding and rationalizing degrader-induced ternary complex formation. Computational models have now started to harness the experimental data from these approaches with the aim to identify and rationally help design new degraders. This review outlines the current experimental and computational strategies used to study ternary complex formation and degradation and highlights the importance of effective crosstalk between these approaches in the advancement of the targeted protein degradation (TPD) field. As our understanding of the molecular features that govern drug-induced interactions grows, faster optimizations and superior therapeutic innovations for TPD and other proximity-inducing modalities are sure to follow.

Baeyer-Villiger monooxygenases (BVMOs) are important flavin-dependent enzymes which perform oxygen insertion reactions leading to valuable products. As reported in many studies, BVMOs are usually unstable during application, preventing a wider usage in biocatalysis. Here, we discovered a novel NADPH-dependent BVMO which originates from Halopolyspora algeriensis using sequence similarity networks (SSNs). The enzyme is stable at temperatures between 10 °C to 30 °C up to five days after the purification, and yields the normal ester product. In this study, the substrate scope was investigated for a broad range of aliphatic ketones and the enzyme was biochemically characterized to identify optimum reaction conditions. The best substrate (86 % conversion) was 2-dodecanone using purified enzyme. This novel BVMO could potentially be applied as part of an enzymatic cascade or in bioprocesses which utilize aliphatic alkanes as feedstock.

Protein tyrosine phosphatases (PTPs) are an important class of enzymes that modulate essential cellular processes through protein dephosphorylation and are dysregulated in various disease states. There is demand for new compounds that target the active sites of these enzymes, for use as chemical tools to dissect their biological roles or as leads for the development of new therapeutics. In this study, we explore an array of electrophiles and fragment scaffolds to investigate the required chemical parameters for covalent inhibition of tyrosine phosphatases. Our analysis juxtaposes the intrinsic electrophilicity of these compounds with their potency against several classical PTPs, revealing chemotypes that inhibit tyrosine phosphatases while minimizing excessive, potentially non-specific reactivity. We also assess sequence divergence at key residues in PTPs to explain their differential susceptibility to covalent inhibition. We anticipate that our study will inspire new strategies to develop covalent probes and inhibitors for tyrosine phosphatases.

Marine bacteria, which are often described as chemical gold, are considered an exceptional source of new therapeutics. Considerable research interest has been given to lipopolysaccharides (LPSs), the main components of the Gram-negative outer membrane. LPS and its lipid A portion from marine bacteria are known to exhibit a tricky chemistry that has been often associated with intriguing properties such as behaving as immune adjuvants or anti-sepsis molecules. In this scenario, we report the structural determination of the lipid A from three marine bacteria within the Cellulophaga genus, which showed to produce an extremely heterogenous blend of tetra- to hexa-acylated lipid A species, mostly carrying one phosphate and one D-mannose on the glucosamine disaccharide backbone. The ability of the three LPSs in activating TLR4 signaling revealed a weaker immunopotential by C. baltica NNO 15840T and C. tyrosinoxydans EM41T , while C. algicola ACAM 630T behaved as a more potent TLR4 activator.

The hydrodynamic effects of macromolecular crowding inside cells are often studied in vitro by using polymers as crowding agents. Confinement of polymers inside cell-sized droplets has been shown to affect the diffusion of small molecules. Here we develop a method, based on digital holographic microscopy, to measure the diffusion of polystyrene microspheres that are confined within lipid vesicles containing a high concentration of solute. We apply the method to three solutes of varying complexity: sucrose, dextran, and PEG, prepared at ∼7 % (w/w). We find that diffusion inside and outside the vesicles is the same when the solute is sucrose or dextran that is prepared below the critical overlap concentration. For poly(ethylene glycol), which is present at a concentration higher than the critical overlap concentration, the diffusion of microspheres inside vesicles is slower, hinting at the potential effects of confinement on crowding agents.

Functional DNA hydrogels with various motifs and functional groups require perfect sequence design to avoid cross-bonding interference with themselves or other structural sequences. This work reports an A-motif functional DNA hydrogel that does not require any sequence design. A-motif DNA is a noncanonical parallel DNA duplex structure containing homopolymeric deoxyadenosines (poly-dA) strands that undergo conformation changes from single strands at neutral pH to a parallel duplex DNA helix at acidic pH. Despite this and other advantages over other DNA motifs like no cross-bonding interference with other structural sequences, the A-motif has not been explored much. We successfully synthesized a DNA hydrogel by using an A-motif as a reversible handle to polymerize a DNA three-way junction. The A-motif hydrogel was initially characterized by electrophoretic mobility shift assay, and dynamic light scattering, which showed the formation of higher-order structures. Further, we used imaging techniques like atomic force microscopy and scanning electron microscope to validating its hydrogel like highly branched morphology. pH-induced conformation transformation from monomers to gel is quick and reversible, and was analysed for multiple acid-base cycles. The sol-to-gel transitions and gelation properties were further examined in rheological studies. The use of the A-motif hydrogel in the visual detection of pathogenic target nucleic acid sequence was demonstrated for the first time in a capillary assay. Moreover, pH-induced hydrogel formation was observed in situ as a layer over the mammalian cells. The proposed A-motif DNA scaffold has enormous potential in designing stimuli-responsive nanostructures that can be used for many biological applications.

Four new CoII complexes, [Co(bpy)2 (acac)]Cl (1), [Co(phen)2 (acac)]Cl (2), [Co(bpy)2 (cur)]Cl (3), [Co(phen)2 (cur)]Cl (4), where bpy=2,2'-bipyridine (1 and 3), phen=1,10-phenanthroline (2 and 4), acac=acetylacetonate (1 and 2), cur=curcumin monoanion (3 and 4) have been designed, synthesized and fully characterized. The X-ray crystal structures of 1 and 2 indicated that the CoN4 O2 core has a distorted octahedral geometry. The photoactivity of these complexes was tuned by varying the π conjugation in the ligands. Curcumin complexes 3 and 4 had an intense absorption band near 435 nm, which made them useful as visible-light photodynamic therapy agents; they also showed fluorescence with λem ≈565 nm. This fluorescence was useful for studying their intracellular uptake and localization in MCF-7 breast cancer cells. The acetylacetonate complexes (1 and 2) were used as control complexes to understand the role of curcumin. The white-light-triggered anticancer profiles of the cytosol targeting complexes 3 and 4 were investigated in detail. These non-dark toxic complexes displayed significant apoptotic photo-cytotoxicity (under visible light) against MCF-7 cells through ROS generation. The control complexes 1 and 2 did not induce significant cell death in the light or dark. Interestingly, 1-4 produced a remarkable antibacterial response upon light exposure. Overall, the reported results here can increase the boundary of the CoII -based anticancer and antibacterial drug development.

Exhausted emission of carbon dioxide (CO2 ) from ships or offshore platforms has become one of the major contributors to global carbon emissions. Enzymes such as carbonic anhydrase (CA) have been widely used for CO2 mineralization because of their high catalytic rate. However, CA in seawater is easy to inactivate and difficult to reuse. Immobilization would be a feasible solution to address the stability issue, which, however, may cause an increase of internal diffusion resistance and reduced catalytic activity. In this regard, design of high-performance biocatalysts for acquiring high catalytic activity and stability of CA is highly desirable. Herein, a monolithic catalyst of Filler-CA@Lys-HOF-1 (FCLH) was prepared by chemical sorption of CA on the surface of the Filler followed by the coating of Lys-HOF-1. The highest catalytic activity of FCLH was obtained by regulating the amount of HOF-1 monomer added. Due to the protection of Lys-HOF-1, the FCLH showed good tolerance against acidity and salinity, which could retain about 80.2 % of the original activity after 9 h incubation in simulated seawater. The catalytic activity of FCLH could retain 85.4 % of the initial activity after 10 cycles. Hopefully, our study can provide a promising biocatalyst for CO2 mineralization, which may drive down carbon emissions when used for CO2 capture and conversion on offshore platforms.

Plants of the genus Allium such as chives, onions or garlic produce S-alk(en)yl cysteine sulfoxides as flavor precursors. Two major representatives are S-propenyl cysteine sulfoxide (isoalliin) and S-propyl cysteine sulfoxide (propiin), which only differ by a double bond in the C3 side chain. The propenyl group of isoalliin is derived from the amino acid valine, but the source of the propyl group of propiin remains unclear. Here, we present an untargeted metabolomics approach in seedlings of chives (Allium schoenoprasum) to track mass features containing sulfur and/or 13 C from labeling experiments with valine-13 C5 guided by their isotope signatures. Our data show that propiin and related propyl-bearing metabolites incorporate carbon derived from valine-13 C5 , but to a much lesser extent than isoalliin and related propenyl compounds. Our findings provide new insights into the biosynthetic pathways of flavor precursors in Allium species and open new avenues for future untargeted labeling experiments.

The transferrin receptor (TfR) mediates transcytosis across the blood-brain barrier (BBB), which offers a promising approach for the non-invasive delivery of therapeutics into the brain parenchyma. Employing the recombinant homodimeric murine TfR ectodomain, prepared in a biochemically functional state, we have selected a cognate Anticalin via phage display and bacterial cell surface display from a random library based on the human lipocalin 2 (Lcn2). After affinity maturation, several engineered lipocalin variants were identified that bind murine TfR in a non-competitive manner with the natural ligand (transferrin ⋅ Fe3+ ), among those an Anticalin - dubbed FerryCalin - exhibiting a dissociation constant (KD ) of 3.8 nM. Epitope analysis using the SPOT technique revealed a sequential epitope in a surface region of TfR remote from the transferrin-binding site. Due to the fast kon rate and short complex half-life, as evidenced by real-time surface plasmon resonance (SPR) measurements, FerryCalin, or one of its related mutants, shows characteristics as a potential vehicle for the brain delivery of biopharmaceuticals.

CRISPR-LbuCas13a has emerged as a revolutionary tool for in vitro diagnosis. Similar to other Cas effectors, LbuCas13a requires Mg2+ to maintain its nuclease activity. However, the effect of other divalent metal ions on its trans-cleavage activity remains less explored. Herein, we addressed this issue by combining experimental and molecular dynamics simulation analysis. In vitro studies showed that both Mn2+ and Ca2+ could replace Mg2+ as cofactors of LbuCas13a. In contrast, Ni2+ , Zn2+ , Cu2+ , or Fe2+ inhibits the cis- and trans-cleavage activity, while Pb2+ does not affect it. Importantly, molecular dynamics simulations confirmed that calcium, magnesium, and manganese hydrated ions have a strong affinity to nucleotide bases, thus stabilizing the conformation of crRNA repeat region and enhancing the trans-cleavage activity. Finally, we showed that combination of Mg2+ and Mn2+ can further enhance the trans-cleavage activity to allow amplified RNA detection, revealing its potential advantage for in vitro diagnosis.

DNA repair proteins participate in extensive protein-protein interactions that promote the formation of DNA repair complexes. To understand how complex formation affects protein function during base excision repair, we used SpyCatcher/SpyTag ligation to produce a covalent complex between human uracil DNA glycosylase (UNG2) and replication protein A (RPA). Our covalent "RPA-Spy-UNG2" complex could identify and excise uracil bases in duplex areas next to ssDNA-dsDNA junctions slightly faster than the wild-type proteins, but this was highly dependent on DNA structure, as the turnover of the RPA-Spy-UNG2 complex slowed at DNA junctions where RPA tightly engaged long ssDNA sections. Conversely, the enzymes preferred uracil sites in ssDNA where RPA strongly enhanced uracil excision by UNG2 regardless of ssDNA length. Finally, RPA was found to promote UNG2 excision of two uracil sites positioned across a ssDNA-dsDNA junction, and dissociation of UNG2 from RPA enhanced this process. Our approach of ligating together RPA and UNG2 to reveal how complex formation affects enzyme function could be applied to examine other assemblies of DNA repair proteins.