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Protein crystallogenesis represents a key step in X‐ray crystallography studies that employ co‐crystallization and ligand soaking for investigating ligand binding to proteins. Co‐crystallization is a method that enables the precise determination of binding positions, although it necessitates a significant degree of optimization. The utilization of microseeding can facilitate a reduction in sample requirements and accelerate the co‐crystallization process. Ligand soaking is the preferred method due to its simplicity; however, it requires careful control of soaking conditions to ensure the successful integration of the ligands. This research protocol details the procedures for co‐crystallization and soaking to achieve protein–ligand complex formation, which is essential for advancing drug discovery. Additionally, a simple protocol for demonstrating soaking for educational purposes is described. Co‐crystallization crystallizes a protein with its ligand, resulting in protein–ligand complex crystals. In contrast, soaking introduces a ligand into preformed protein crystals, allowing it to bind. Both methods produce crystals for X‐ray diffraction, which generates diffraction patterns that are analyzed to determine the three‐dimensional structure of the complex. This process uncovers key interactions critical to understanding the protein's biological functions.

The global accumulation of plastic waste, exceeding 360 million tonnes annually, represents a critical environmental challenge due to their widespread use and extreme recalcitrance in natural environments. Furthermore, the end‐of‐life processing of bioplastics, which are often marketed as eco‐friendly, remains problematic, with biodegradation often requiring industrial conditions. Enzyme‐based depolymerization of polyesters, such as polyethylene terephthalate (PET) and bioplastics (e.g., polylactic acid (PLA), poly(butylene adipate‐co‐terephthalate) (PBAT), and polyhydroxyalkanoates (PHAs)), has emerged as a promising alternative, offering a green approach to postconsumer plastic management with a reduced environmental impact and in alignment with circular economy principles. This review summarizes recent advances in enzymatic degradation of oil‐derived and bio‐based polyesters. Key recent developments are discussed including novel high‐throughput screenings, computational workflow for improvement of PET hydrolases and de novo design of biocatalysts, microbial platforms, and enzyme‐embedded self‐biodegrading bioplastics. Collectively, these innovations are redefining the role of biocatalysis in tackling synthetic polymer pollution. Looking ahead, the integration of enzymatic depolymerization with upcycling pathways, standardized kinetic metrics, and one‐pot bioprocesses represents a viable strategy for sustainable plastic waste valorization. Plastic pollution remains a critical environmental challenge, and current mechanical and chemical recycling methods are insufficient to achieve a fully circular economy. This review highlights recent breakthroughs in the enzymatic depolymerization of both oil‐derived polyesters and bioplastics, including high‐throughput protein engineering, de novo enzyme design, microbial recycling platforms, and enzyme‐embedded self‐degrading materials.

The production of high‐quality crystals is a key step in crystallography in general, but control of crystallization conditions is even more crucial in serial crystallography, which requires sets of crystals homogeneous in size and diffraction properties. This protocol describes the implementation of a simple and user‐friendly microfluidic device that allows both the production of crystals by the counter‐diffusion method and their in situ analysis by serial crystallography. As an illustration, the whole procedure is used to determine the crystal structure of three proteins from data collected at room temperature at a synchrotron radiation source. This protocol describes the implementation of a simple and user‐friendly microfluidic device called CrystalChip that allows the production of crystals by the counter‐diffusion method and their in situ analysis by serial crystallography. As an illustration, it is applied to determine the 3D structure of three proteins from data collected at room temperature at a synchrotron radiation source.

Cell‐free DNA (cfDNA) in blood plasma can be bound to nucleosomes that contain post‐translational modifications representing the epigenetic profile of the cell of origin. This includes histone H3 lysine 36 trimethylation (H3K36me3), a marker of active transcription. We hypothesised that cell‐free chromatin immunoprecipitation (cfChIP) of H3K36me3‐modified nucleosomes present in blood plasma can delineate tumour gene expression levels. H3K36me3 cfChIP followed by targeted NGS (cfChIP‐seq) was performed on blood plasma samples from non‐small‐cell lung cancer (NSCLC) patients (NSCLC, n = 8), small‐cell lung cancer (SCLC) patients (SCLC, n = 4) and healthy controls (n = 4). H3K36me3 cfChIP‐seq demonstrated increased enrichment of mutated alleles compared with normal alleles in plasma from patients with known somatic cancer mutations. Additionally, genes identified to be differentially expressed in SCLC and NSCLC tumours had concordant H3K36me3 cfChIP enrichment profiles in NSCLC (sensitivity = 0.80) and SCLC blood plasma (sensitivity = 0.86). Findings here expand the utility of cfDNA in liquid biopsies to characterise treatment resistance, cancer subtyping and disease progression. Blood plasma contains H3K36me3‐modified nucleosomes representing active genes in the cell of origin. Our findings show that cell‐free chromatin immunoprecipitation (cfChIP) of H3K36me3‐nucleosomes in plasma from lung cancer patients can reveal tumour gene expression. cfChIP followed by next‐generation sequencing identifies genes that are differentially expressed in the tumour of non‐small‐cell and small‐cell lung cancer patients. Created with BioRender.com.

Frailty, a reversible clinical geriatric syndrome, impairs the ability to maintain homeostasis, leading to severe consequences such as hospitalization and death. Cognitive frailty, characterized by the co‐occurrence of physical frailty and cognitive impairment, has garnered increasing attention in recent years. Preclinical models, especially rodent studies, are essential for understanding frailty and developing interventions to mitigate associated conditions. Traditionally, animal studies have focused solely on physical frailty. We have pioneered the inclusion of cognitive parameters by developing a novel physical‐cognitive frailty score (FS) in animal research, in order to assess the effectiveness of anti‐aging interventions. Here, we provide a detailed example of the FS calculation at the group level, which can serve as a guide for other studies. This dual‐focus approach also helps in understanding how physical frailty and cognitive impairment interact to exacerbate adverse health outcomes and provides an opportunity to evaluate potential interventions that target both physical and cognitive dimensions of frailty more reliably. Method for Frailty Score (FS) calculation: (1) Combine all animals that will be compared in one group; (2) Calculate 20% cutoff; (3) Determine the number of animals that ‘failed’ or ‘passed’; (4) Calculate ‘percentage of success’ for graphical representation of parameters; (5) Repeat for all parameters; (6) Calculate FS and use percentages for graphical representation of FS.

Macroautophagy/autophagy is a crucial cellular process for degrading and recycling damaged proteins and organelles, playing a significant role in diseases such as cancer and neurodegeneration. Evaluating autophagy flux, which tracks autophagosome formation, maturation, and degradation, is essential for understanding disease mechanisms. Current fluorescence‐based methods are resource‐intensive, requiring advanced equipment and expertise, limiting their use in clinical laboratories. Here, we introduce a non‐fluorescent immunohistochemistry (IHC) method using MAP1LC3/LC3 and SQSTM1 as core markers for autophagy flux assessment. LC3 levels reflect autophagosome formation, whereas SQSTM1 degradation and a decrease in the number of its puncta indicate active flux (i.e., lysosomal turnover). We optimized chromogenic detection using diaminobenzidine (DAB) staining and developed a scoring system based on puncta number and the percentage of stained cells. This accessible, cost‐effective method enables reliable autophagy quantification using a standard light microscope, bridging the gap between experimental research and clinical diagnostics. Our protocol allows accurate autophagy evaluation in fixed tissues, offering practical applications in biomedical research and clinical pathology assessment. We introduce an immunohistochemistry method to measure autophagy flux, highlighting the active degradation and recycling of cellular waste. This cost‐effective approach uses tissue samples to track key markers like LC3 and SQSTM1, revealing how cells maintain health or respond to diseases such as cancer. It bridges the gap between research and clinical applications, offering practical diagnostic and therapeutic insights.

The functional characterization of plasma membrane transport proteins often relies on their heterologous expression in cultured cells. However, some transporters exhibit low activity, hindering meaningful functional assays. Heterologous expression is usually based on strong viral promoters which in living cells are prone to promoter silencing, a major problem. Here, we investigated the efficacy of low‐cost histone deacetylase (HDAC) inhibitors in enhancing transporter activity, comparing the established sodium butyrate (the sodium salt of butyric acid) with valproate/valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA, also known as vorinostat). Using 293 cells stably transfected with pEBTet plasmids containing the CMV promotor to express the transporters SLC16A9, SLC22A15, and OATP1A2, we measured substrate efflux or uptake via LC–MS/MS following overnight preincubation with the HDAC inhibitors. All three compounds markedly stimulated transporter activity. VPA was less effective than butyrate but still surpassed control conditions. SAHA was cytotoxic at 6 μm, but at 2 μm, the enhancement was consistently comparable to 5 mm butyrate. Additionally, SAHA was more cost‐effective and devoid of the repulsive odor characteristic of butyrate. Our findings advocate for replacing butyrate with SAHA to enhance heterologously expressed transporter activity. This offers a more efficient and user‐friendly alternative for functional assays. Heterologous expression of membrane transporters in cultured cells is essential for functional characterization, but is sometimes limited by low activity. Our study compares the HDAC inhibitors butyrate, VPA and SAHA to enhance transport activity. We propose to replace butyrate by SAHA: it is equally effective, devoid of repulsive odor, costs less, and the required concentration is 2500 times lower.

Crystallization of recombinant proteins in living cells is an emerging approach complementing conventional crystallization techniques. Homogeneous microcrystals well suited for serial diffraction experiments at X‐ray free‐electron lasers and synchrotron sources can be produced in a quasi‐native environment, without the need for target protein purification. Several protein structures have already been solved; however, exploiting the full potential of this approach requires a systematic and versatile screening strategy for intracellular crystal growth. Recently, we published InCellCryst, a streamlined pipeline for producing microcrystals within living insect cells. Here, we present the detailed protocol, including optimized target gene expression using a baculovirus vector system, crystal formation, detection, and serial X‐ray diffraction directly in the cells. The specific environment within the different cellular compartments acts as a screening parameter to maximize the probability of crystal growth. If successful, diffraction data can be collected 24 days after the start of target gene cloning. This research protocol provides a step‐by‐step guide for applying the InCellCryst pipeline for intracellular protein crystallization. After gene cloning and generation of recombinant baculoviruses, High Five insect cells are infected for target protein crystallization. If detection is successful, the crystal‐containing cells are mounted for serial X‐ray diffraction data collection directly in the cells, followed by data processing and structure elucidation.

Recombinant protein production in Escherichia coli is a fundamental aspect of biotechnology. Fusion tags are commonly used to enhance solubility and facilitate purification. However, these tags can lead to challenges such as low yields, complicated purification processes, and the necessity for tag removal, especially when dealing with peptides. This study introduces a novel fusion tag called SmallTalk, a truncated version of the small metal‐binding protein SmbP. Weighing in at 5 kDa, SmallTalk includes two of the four α‐helices found in SmbP. It retains the ability to bind Ni(II) ions, which enables purification through IMAC. In this work, we assessed the efficiency of SmallTalk in expressing and purifying both a model protein, the green fluorescent protein, and the antimicrobial peptide Bin1b. Both proteins were effectively expressed and purified using IMAC, demonstrating SmallTalk's value as an affinity tag, yielding 7.2 mg·L−1 of cell culture for the green fluorescent protein and up to 9.8 mg·L−1 for Bin1b. Antimicrobial assays conducted with SmallTalk‐tagged Bin1b showed activity against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, with minimum inhibitory concentrations ranging from 7.5 to 22.5 μm. Importantly, SmallTalk enabled the full retention of Bin1b's antimicrobial activity without the need for its removal, significantly simplifying the production process. These findings indicate that SmallTalk provides a promising strategy for the recombinant production of peptides. This tag has the potential to enhance the expression, purification, and functional analysis of antimicrobial peptides, which are increasingly being pursued as alternatives to antibiotics in the fight against antimicrobial resistance. The SmallTalk fusion tag allows for the efficient expression and purification of soluble recombinant proteins or peptides in Escherichia coli. Testing with SmallTalk‐GFP confirmed that the proteins were soluble and folded correctly, while SmallTalk‐Bin1b maintained its antimicrobial activity against various bacterial isolates. This streamlined workflow showcases the versatility of the SmallTalk system in producing functional, biologically active proteins and peptides.

Intracellular Ca2+ regulates insulin secretion from pancreatic β‐cells and is influenced by cannabinoid signaling. However, the hydrophobicity and complex pharmacology of cannabinoid ligands prevent precision receptor targeting, limiting our understanding of their roles in modulating insulin release. Here, we use fluorescent Ca2+ imaging to examine how the light‐activatable CB2 receptor agonist azo‐HU308 modulates Ca2+ dynamics in INS‐1 β‐cells. UV‐A photoactivation of azo‐HU308 triggered robust, repeatable Ca2+ transients, and pharmacological profiling revealed this effect was independent of CB2 receptor activation but was instead mediated by extracellular Ca2+ influx through TRPC channels. These findings position azo‐HU308 as a novel optical tool for controlling β‐cell Ca2+ levels and highlight a non‐GPCR pathway by which synthetic cannabinoids can modulate Ca2+ dynamics in excitable cells. Light activation of the photoswitchable cannabinoid ligand azo‐HU308 triggers Ca2+ influx in pancreatic β‐cells through TRPC channels, independent of CB2 cannabinoid receptors. This reveals a non‐GPCR pathway for cannabinoid modulation of β‐cell Ca2+ dynamics and establishes azo‐HU308 as an optical tool to study cannabinoid signaling through TRP channels in excitable cells.