Cultivated meat (CM) is an emerging field of research that applies cell-culture and bioprocessing technologies to the production of animal-derived food products without conventional animal farming. Growing global demand for meat, coupled with environmental and resource constraints associated with existing production systems, has motivated increasing academic, industrial, and policy interest in CM. Beyond its research and commercial implications, CM has the potential to contribute to social stability and food security, particularly in regions with limited arable land and high dependence on food imports, and may reduce vulnerability to global disruptions such as zoonotic disease outbreaks (e.g., avian influenza). This review offers a comprehensive analysis of the field, addressing the growing demand for CM, technical advancements, and the challenges in translating CM production into scalable applications. It examines current technological obstacles, highlights recent research progress, and explores potential solutions for achieving sustainable growth in the industry. By evaluating the intricacies of CM, this review aims to provide insights into the strategies needed to advance this innovative field and meet future demands for sustainable and secure food sources.

Protein phosphorylation is one of the most common and versatile regulatory mechanisms in cells. Most human proteins are phosphorylated at multiple sites, giving rise to large numbers of possible phosphorylation patterns. Each phosphorylation pattern can lead to a different functional or pathological outcome. Yet, linking defined phosphorylation patterns to specific biological functions remains a major experimental challenge. In this review we describe the main strategies to study phosphorylation patterns at the protein and domain levels and highlight how they complement each other. We first discuss cellular approaches, including phosphomimetics, kinase-based assays, and genetic code expansion, which allow working in a native environment but have their significant drawbacks. We then describe in vitro methods, such as enzymatic phosphorylation and semi-synthetic phosphoproteins generated by ligation, which afford mechanistic insights but result in low yields and are difficult to scale for producing libraries. We focus on synthetic phosphopeptide libraries as tools that offer precise control over the number and position of phosphosites and are uniquely suited for systematic mapping of phosphorylation patterns. This comes at a price of not working at the protein level, but rather at the domain level. Peptide libraries are often used for preliminary identification of key phosphorylations, later studied in detail at the protein level. We conclude that ideally more than one method should be used and that these methods should not be viewed as competing but rather as complementary. A combined use of several of these approaches provides a practical toolbox for dissecting how phosphorylation patterns regulate protein behavior.

Glycosylation is a post-translational modification prevalent in the majority of proteins. Many glycoproteins contain several glycosylation sites, often bearing different glycan moieties. The inherent difficulties of glycopeptide synthesis worsen for heterogeneously glycosylated peptides, as each glycan introduces unique synthetic hurdles. Stirring-assisted solid-phase synthesis proved extremely valuable in accessing post-translational modified peptides. We present the stirring-assisted synthesis of a heterogeneous glycopeptide library, derived from α-Dystroglycan, bearing a variety of glycosylation patterns combining both mannose and GalNAc cores. The developed strategy streamlined the expeditious assembly with the post-assembly manipulation, enabling the procurement of heterogeneously glycosylated peptides in high purity.

 

Rett syndrome (RTT) is an X-linked neurodevelopment disorder associated with the single monogenic mutation in methyl-CpG binding protein 2 (MeCP2) in up to 95% of cases. The growing number of genome-wide association studies and incomplete concordance for autoimmune diseases in monozygotic twins concur to support the involvement of environmental factors, like infectious agents or chemicals, in the breakdown of tolerance leading to autoimmunity. In fact, the coexistence of a dysregulation of the immune system in RTT patients has been previously hypothesized. We herein explored the hypothesis that an autoimmune component derived from environmental bacterial infection of non-typeable Haemophilus influenzae may coexist in RTT. At this purpose we screened sera from RTT syndrome patients, non-RTT pervasive developmental disorders patients and healthy controls with the hyper-glucosylated adhesin protein HMW1Ct-Glc used as an antigen in ELISA in order to identify specific antibodies to N-glucosylation sites. Results showed that HMW1Ct-Glc is able to significantly discriminate antibodies among RTT sera and controls. Competitive ELISA confirmed the specific interaction between antibodies characteristic of RTT syndrome and the N-glucosylation motifs of the bacterial adhesin protein HMW1Ct-Glc. 

Glycosylation is among the most common posttranslational
modifications of proteins. There is a great synthetic and
practical difficulty in the assembly and deprotection of glycopeptides.
State-of-the-art methods for glycopeptide synthesis are wasteful of
glycosylated amino acids, are slow, and suffer from low yields. These
shortcomings hamper accessibility to multiply glycosylated peptides.
We report the accelerated, high-shear stirring-assisted synthesis of
multiply O-glycosylated peptides. The equimolar assembly was
streamlined with deacetylation to provide multiglycosylated peptides
at high purity. Cadherin-derived multiglycosylated peptides synthesized
in large quantities provided selective Listeria monocytogenes
electrochemical biosensing.

Semiconducting single-walled carbon nanotubes (sc-SWCNTs) are of great potential for vapor sensing. However, sc-SWCNTs lack recognition features for discriminating between sparsely functionalized moieties, molecules with similar structural features, and enantiomer pairs. This becomes a major setback in discriminating between volatile organic compounds (VOCs). Here, we used two galactosides decorated with aromatic groups as a recognition layer in chemoresistive sc-SWCNT sensors to produce chiral preference toward six terpenoid enantiomers. The multichirality and multifunctionality of a monosaccharide scaffold were exploited to maximize the limited interacting features associated with VOCs. The developed system establishes a robust and tunable platform for enantioselective gas sensing.

We introduce the MORE-Q dataset, a quantum-mechanical (QM) dataset encompassing the structural and electronic data of non-covalent molecular sensors formed by combining 18 mucin-derived olfactorial receptors with 102 body odor volatilome (BOV) molecules. To have a better understanding of their intra- and inter-molecular interactions, we have performed accurate QM calculations in different stages of the sensor design and, accordingly, MORE-Q splits into three subsets: i) MORE-Q-G1: QM data of 18 receptors and 102 BOV molecules, ii) MORE-Q-G2: QM data of 23,838 BOV-receptor configurations, and iii) MORE-Q-G3: QM data of 1,836 BOV-receptor-graphene systems. Each subset involves geometries optimized using GFN2-xTB with D4 dispersion correction and up to 39 physicochemical properties, including global and local properties as well as binding features, all computed at the tightly converged PBE+D3 level of theory. By addressing BOV-receptor-graphene systems from a QM perspective, MORE-Q can serve as a benchmark dataset for state-of-the-art machine learning methods developed to predict binding features. This, in turn, can provide valuable insights for developing the next-generation mucin-derived olfactory receptor sensing devices.

Cholera is a severe infectious disease caused by Vibrio cholerae. The disease primarily spreads through contaminated food and water sources; it remains a significant global health concern. The pathogenesis of Vibrio cholerae is facilitated by the secreted neuraminidase, Vibrio cholerae neuraminidase (VCNA). This neuraminidase cleaves host cell surface sialic acids, which leads to bacterial colonization and infection progression. This study presents the development of a label-free VCNA biosensor based on electrochemical impedance spectroscopy. The biosensor relies on synthetic sialosides that form self-assembled monolayers on gold electrodes. The system demonstrated selective detection of VCNA activity through distinct impedance variations corresponding to the enzymatic cleavage of the sialoside substrates. The VCNA activity was evaluated under varying environmental conditions, including different media and pH values. This approach provides insights into developing robust biosensing platforms for bacterial detection, offering potential applications in various diagnostic and monitoring systems.

Optimization of glycosylation temperatures is essential for automated solid phase glycan assembly. Constructing a reliable optimization system that mimics key reaction components is crucial for increasing the atom economy and the energy efficiency of the process. Although glycosyl acceptors play a pivotal role in glycosylation, evaluating their effect on glycosylation efficiency is nontrivial. While screening various glycosyl acceptors for optimization is impractical, compromising for simple alkyl acceptors produces results of dubious relevance. We demonstrate that optimization with a modified trans-4-aminocyclohexanol-based acceptor accurately reflects the optimal glycosylation temperature of several glycosyl acceptors, overcoming previous limitations. The contextual use of both n-alkyl and glycan-mimetic acceptors for automated optimization enabled translation of optimized glycosylation temperature to highly efficient solid phase synthesis of disaccharides.

Specific phosphorylation patterns regulate the activity of proteins and play a central role in protein self-assembly. In Tau, such patterns drive the formation of disease-related condensates and aggregates. Understanding their functional impact is essential for studying Tauopathies such as Alzheimer’s Disease. Here we show how specific phosphorylation patterns regulate Tau self-assembly and control the interplay between its aggregation and condensation, using a peptide-based approach that allows systematic analysis of libraries of specific phosphorylation patterns at the domain level and is complementary to the current protein-level methods. We applied our methodology to study the effect of specific phosphorylations on the aggregation and condensation of the R4 domain of Tau that is pivotal for its self-assembly, forming the β-helix motif that is common to various Tau patient fibrils. Using advanced phosphopeptide synthesis methods developed in our labs, we generated a library of multi-phosphorylated peptides derived from Tau R4. We found that phosphorylation at Ser341 promotes aggregation, while Ser352 enhances condensation. Phosphorylation at Ser356 inhibits both processes. The source of these different outcomes is the distinct microenvironments around each phosphorylated site. Our results provide a residue-level resolution of how the decision between Tau condensation and aggregation is being made. This was possible by using our peptide-based approach, which is complementary to protein-level method and enables efficient identification of active phosphorylation patterns. These can later be studied at the protein level.

Heparan sulfate (HS) interactions with interleukin 8 (IL-8) are crucial for immune system response. The structural features of the HS and the environmental entities, such as metal ions, can regulate these interactions. However, it is challenging to evaluate the effect of each parameter on the interactions because of low accessibility to well-defined saccharides and the lack of characteristic features to be determined by analytical tools. We evaluated the effect of the HS structural features on IL-8 binding affinity utilizing electrochemical impedance spectroscopy (EIS) and X-ray photoelectron spectroscopy (XPS). We showed that the metal ions Ca²⁺ and Mg²⁺ dissimilarly mediate the interactions of HS and IL-8 in structure-dependent manner of the HS. We showed that in all glycans, a positive synergistic effect on IL-8 binding was observed. For several glycans, the presence of ions resulted in a dramatic increase in the affinity to IL-8, while for other glycans, a milder effect was observed. This demonstrated that both structural motifs and environmental features are crucial for maintaining the interactions between the HS and IL-8.

Streptococcus pneumoniae is a pathogenic bacterium that contains the surface-bound neuraminidase, NanA. NanA has two domains that interact with sialosides. It is hard to determine the contribution of each domain separately on catalysis or binding. In this work, we used biochemical methods to obtain the separated domains, applied electrochemical and surface analysis approaches, and determined the catalytic and binding preferences toward a surface-bound library of sialosides. Impedimetric studies on two different surfaces revealed that protein–surface interactions provide a tool for distinguishing the unique contribution of each domain at the interface affecting the substrate preference of the enzyme in different surroundings. We showed that each domain has a sialoside-specific affinity. Furthermore, while the interaction of the sialoside-covered surface with the carbohydrate-binding domain results in an increase in impedance and binding, the catalytic domain adheres to the surface at high concentrations but retains its catalytic activity at low concentrations.

Synthesis of complex glycans is extremely challenging. In the current issue of Device, Seeberger and co-workers introduce a new oligosaccharide synthesizer. The introduced technology integrates innovative solutions to overcome difficulties associated with solidphase oligosaccharide synthesis. The focus on an energy efficient, smaller, and user-friendly device forecasts exciting advances in glycochemistry.

Neuraminidases (NA) are sialic acid-cleaving enzymes that are used by both bacteria and viruses. These enzymes have sialoside structure-related binding and cleaving preferences. Differentiating between these enzymes requires using a large array of hard-to-access sialosides. In this work, we used electrochemical impedimetric biosensing to differentiate among several pathogene-related NAs. We used a limited set of sialosides and tailored the surface properties. Various sialosides were grafted on two different surfaces with unique properties. Electrografting on glassy carbon electrodes provided low-density sialoside-functionalized surfaces with a hydrophobic submonolayer. A two-step assembly on gold electrodes provided a denser sialoside layer on a negatively charged submonolayer. The synthesis of each sialoside required dozens of laborious steps. Utilizing the unique protein–electrode interaction modes resulted in richer biodata without increasing the synthetic load. These principles allowed for profiling NAs and determining the efficacy of various antiviral inhibitors.

Sialic acid recognition and hydrolysis are essential parts of cellular function and pathogen infectivity. Neuraminidases are enzymes that detach sialic acid from sialosides, and their inhibition is a prime target for viral infection treatment. The connectivity and type of sialic acid influence the recognition and hydrolysis activity of the many different neuraminidases. The common strategies to evaluate neuraminidase activity, recognition, and inhibition rely on extensive labeling and require a large amount of sialylated glycans. The above limitations make the effort of finding viral inhibitors extremely difficult. We used synthetic sialylated glycans and developed a label-free electrochemical method to show that sialoside structural features lead to selective neuraminidase biosensing. We compared Neu5Ac to Neu5Gc sialosides to evaluate the organism-dependent neuraminidase selectivity–sensitivity relationship. We demonstrated that the type of surface and the glycan monolayer density direct the response to either binding or enzymatic activity. We proved that while the hydrophobic glassy carbon surface increases the interaction with the enzyme hydrophobic interface, the negatively charged interface of the lipoic acid monolayer on gold repels the protein and enables biocatalysis. We showed that the sialoside monolayers can serve as tools to evaluate the inhibition of neuraminidases both by biocatalysis and molecular recognition.

Design and synthesis of orthogonally protected monosaccharide building blocks are crucial for the preparation of well-defined oligosaccharides in a stereo- and regiocontrolled manner. Selective introduction of protecting groups to partially protected monosaccharides is nontrivial due to the often unpredictable electronic, steric, and conformational effects of the substituents. Abolished reactivity toward a commonly used Lewis base-catalyzed acylation of O-2 was observed in conformationally restricted 4,6-O-benzylidene-3-O-Nap galactoside. Investigation of analogous systems, crystallographic characterization, and quantum chemical calculations highlighted the overlooked conformational and steric considerations, the combination of which produces a unique passivity of the 2-OH nucleophile. Evaluating the role of electrophile counterion and auxiliary base in the acylation of the sterically crowded and conformationally restricted galactoside system revealed an alternative Brønsted base-driven reaction pathway via nucleophilic activation. Insights gained from this model system were utilized to access the target galactoside intermediate within the envisioned synthetic route. The acylation strategy described herein can be implemented in future syntheses of key monomeric building blocks with unique protecting group hierarchies.

Solid phase synthesis is the most dominant approach for the preparation of biological oligomers as it enables the introduction of monomers iteratively. Accelerated solid phase synthesis of biological oligomers is crucial for chemical biology, but its application to the synthesis of oligosaccharides is not trivial. Solid-phase oligosaccharide assembly is a slow process performed in a variety of conditions and temperatures, requires an inert gas atmosphere, and demands high excess of glycosyl donors. The process is done in special synthesizers and poor mixing of the solid support increases the risk of diffusion-independent hydrolysis of the activated donors. High shear stirring is a new way to accelerate solid phase synthesis. The efficient mixing ensures that reactive intermediates can diffuse faster to the solid support thereby increasing the kinetics of the reactions. We report here a stirring-based accelerated solid-phase oligosaccharide synthesis. We harnessed high shear mixing to perform diffusion-dependent glycosylation in a short reaction time. We minimized the use of glycosyl donors and the need to use an inert atmosphere. We showed that by tailoring the deprotection and glycosylation conditions to the same temperature, assembly steps are performed continuously, and full glycosylation cycles is completed in minutes.

The biosensing of bacterial pathogens is of a high priority. Electrochemical biosensors are an important future tool for rapid bacteria detection. A monolayer of bacterial-binding peptides can serve as a recognition layer in such detection devices. Here, we explore the potential of random peptide mixtures (RPMs) composed of phenylalanine and lysine in random sequences and of controlled length, to form a monolayer that can be utilized for sensing. RPMs were found to assemble in a thin and diluted layer that attracts various bacteria. Faradaic electrochemical impedance spectroscopy was used with modified gold electrodes to measure the charge-transfer resistance (RCT) caused due to the binding of bacteria to RPMs. Pseudomonas aeruginosa was found to cause the most prominent increase in RCT compared to other model bacteria. We show that the combination of highly accessible antimicrobial RPMs and electrochemical analysis can be used to generate a new promising line of bacterial biosensors.

 

Peptide fragments of glycoproteins containing multiple N-glycosylated sites are essential biochemical tools not only to investigate protein–protein interactions but also to develop glycopeptide-based diagnostics and immunotherapy. However, solid-phase synthesis of glycopeptides containing multiple N-glycosylated sites is hampered by difficult couplings, which results in a substantial drop in yield. To increase the final yield, large amounts of reagents but also time-consuming steps are required. Therefore, we propose herein to utilize heating and stirring in combination with low-loading solid supports to set up an accelerated route to obtain, by an efficient High-Temperature Fast Stirring Peptide Synthesis (HTFS-PS), glycopeptides containing multiple N-glycosylated sites using equimolar excess of the precious glycosylated building blocks.

 

Optimizing glycosylation conditions for automated glycan assembly is highly challenging, demand wasteful use of precious building blocks and rely on nontrivial analyses. We developed a semi-quantitative method for automated optimization of glycosylation temperature that utilized minute quantities of donors and translated those conditions to solid-phase glycan assembly.