Search Results (210)

Conventional methods for generating knock-out or knock-in mammalian cell models using CRISPR-Cas9 genome editing often require tedious single-cell clone selection and expansion. In this study, we develop and optimise rapid and robust strategies to engineer homozygous fluorescent reporter knock-in cell pools with precise genome editing, circumventing clonal variability inherent to traditional approaches. To reduce false-positive cells associated with random integration, we optimise the design of donor DNA by removing the start codon of the fluorescent reporter and incorporating a self-cleaving T2A peptide system. Using fluorescence-assisted cell sorting (FACS), we efficiently identify and isolate the desired homozygous fluorescent knock-in clones, establishing stable cell pools that preserve parental cell line heterogeneity and faithfully reflect endogenous transcriptional regulation of the target gene. We evaluate the knock-in efficiency and rate of undesired random integration in the electroporation method with either a dual-plasmid system (sgRNA and donor DNA in two separate vectors) or a single-plasmid system (sgRNA and donor DNA combined in one vector). We further demonstrate that coupling our single-plasmid construct with an integrase-deficient lentivirus vector (IDLV) packaging system efficiently generates fluorescent knock-in reporter cell pools, offering flexibility between electroporation and lentivirus transduction methods. Notably, compared to the electroporation methods, the IDLV system significantly minimises random integration. Moreover, the resulting reporter cell lines are compatible with most of the available genome-wide sgRNA libraries, enabling unbiased CRISPR screens to identify key transcriptional regulators of a gene of interest. Overall, our methodologies provide a powerful genetic tool for rapid and robust generation of fluorescent reporter knock-in cell pools with precise genome editing by CRISPR-Cas9 for various research purposes. Full article

Stem cells, unspecialized cells with regenerative and differentiation capabilities, hold immense potential in regenerative medicine, exemplified by hematopoietic stem cell transplantation. However, their clinical application faces significant limitations, including their tumorigenic risk due to uncontrolled proliferation and cellular heterogeneity. This review explores how synthetic biology, an interdisciplinary approach combining engineering and biology, offers promising solutions to these challenges. It discusses the concepts, toolkit, and advantages of synthetic biology, focusing on the design and integration of genetic circuits to program stem cell differentiation and engineer safety mechanisms like inducible suicide switches. This review comprehensively examines recent advancements in synthetic biology applications for stem cell engineering, including programmable differentiation circuits, cell reprogramming strategies, and therapeutic cell engineering approaches. We highlight specific examples of genetic circuits that have been successfully implemented in various stem cell types, from embryonic stem cells to induced pluripotent stem cells, demonstrating their potential for clinical translation. Despite these advancements, the integration of synthetic biology with mammalian cells remains complex, necessitating further research, standardized datasets, open access repositories, and interdisciplinary collaborations to build a robust framework for predicting and managing this complexity. Full article

Plant-derived vesicle-like nanoparticles (PDVLNs) are bioactive nanovesicles secreted by plant cells, emerging as a novel therapeutic tool for tissue repair and regeneration due to their low immunogenicity, intrinsic bioactivity, and potential as drug delivery carriers. This review examines PDVLNs’ biogenesis mechanisms, isolation techniques, and compositional diversity, emphasizing their roles in promoting essential regenerative processes—cell proliferation, differentiation, migration, immune modulation, and angiogenesis. We explore their therapeutic applications across multiple tissue types, including skin, bone, neural, liver, gastrointestinal, cardiovascular, and dental tissues, using both natural and engineered PDVLNs in various disease models. Compared to mammalian exosomes, PDVLNs offer advantages such as reduced immune rejection and ethical concerns, enhancing their sustainability and appeal for regenerative medicine. However, challenges in clinical translation, including scalability, standardization, and safety remain. This paper consolidates current knowledge on PDVLNs, highlighting their versatility and providing insights into engineering strategies to optimize efficacy, ultimately outlining future research directions to advance their clinical potential. Plant vesicle-like nanoparticles (PDVLNs) may become a new avenue for the treatment of tissue injury, promoting tissue repair and regeneration through their intrinsic bioactivity or as drug delivery carriers. In addition, PDVLNs can be engineered and modified to achieve better results. Full article

DNA is inherently unstable and is susceptible to damage from both endogenous sources (such as reactive oxygen species) and exogenous factors (including UV, ionizing radiation, and chemicals). The accumulation of DNA damage manifests as genetic mutations, chromosomal instability, and the stalling of DNA replication and transcription processes. Accumulated DNA damage influences apoptosis and cell cycle checkpoints, serving as one of the key triggers for the manifestation of the senescent phenotype. Both aging and cancer are associated with the accumulation of mutations in somatic cells. Disruption of cell cycle control and uncontrolled proliferation are fundamental characteristics of any cancer cell, with the majority of anticancer drugs acting as inhibitors of cyclin-dependent kinases, thereby inducing a transition of cells into a senescent state. Consequently, disturbances in the dynamics and regulation of inflammatory responses, oxidative stress, cell proliferation, DNA damage repair, and epigenetic anomalies, along with the influence of retroviruses and transposons, lead to the accumulation of senescent cells within the human body, characterized by blocked replication and cell cycle, as well as a distinct secretory phenotype. The age-related or disease-associated accumulation of these senescent cells significantly alters the physiology of tissues and the organism as a whole. Many secondary metabolites of higher plants exhibit senolytic and senomorphic activities, although most of them are not fully characterized. In this review, we will explore the principal signaling pathways in mammalian cells that govern the cell cycle and cellular senescence, with a particular emphasis on how their dynamics, expression, and regulation have been modified through the application of senotherapeutic compounds. The second section of the review will identify key target genes for the metabolic engineering, primarily aimed at enhancing the accumulation of plant secondary metabolites with potential therapeutic benefits. Lastly, we will discuss the rationale for utilizing liver cells as a model system to investigate the effects of senolytic compounds on human physiology and health, as well as how senotherapeutic substances can be leveraged to improve gene therapy approaches based on CRISPR/Cas9 and prime-editing technologies. Full article

In this work, we present an innovative, crosslinker-free method for preparing chitosan-based hydrogel precursors, fully aligned with green chemistry principles and composed of only five non-toxic, readily available reagents. The key novelty lies in the use of glycerin, which, during thermal annealing, evaporates and triggers a surface or bulk chemical transformation of chitosan, depending on its concentration. This process significantly enhances the material’s mechanical properties after swelling—with up to a 35% increase in tensile strength and a notable reduction in water uptake compared to systems containing AMPS-based crosslinkers. FTIR analysis indicates a partial re-acetylation of chitosan, shifting its structure toward that of chitin, which correlates with improved hydrophobicity (as shown by increased contact angles up to 92°) and greater structural integrity. These improvements are particularly pronounced at glycerin concentrations of 10–20%, whereas higher concentrations (50%) result in brittle, non-moldable films. Importantly, preliminary biological tests confirm that the resulting hydrogels are effectively colonized by mammalian cells, making them promising candidates for bioimplant or tissue engineering applications. Surface morphology and compatibility were further assessed via SEM, AFM, and contact angle measurements. Full article

DNA methylation, a cornerstone of epigenetic regulation, governs critical biological processes including transcriptional modulation, genomic imprinting, and transposon suppression through chromatin architecture remodeling. Recent advances have revealed that aberrant methylation patterns—characterized by spatial-temporal dysregulation and stochastic molecular noise—serve as key drivers of diverse pathological conditions, from oncogenesis to neurodegenerative disorders. However, the field faces dual challenges: (1) current understanding remains fragmented due to the inherent spatiotemporal heterogeneity of methylation landscapes across tissues and developmental stages, and (2) mechanistic insights into non-canonical methylation pathways (particularly 6mA) in non-mammalian systems are conspicuously underdeveloped. This review systematically synthesizes the evolutionary-conserved versus species-specific features of 5-methylcytosine (5mC) and N6-methyladenine (6mA) regulatory networks across three biological kingdoms. Through comparative analysis of methylation/demethylation enzymatic cascades (DNMTs/TETs in mammals, CMTs/ROS1 in plants, and DIM-2/DNMTA in fungi), we propose a unified framework for targeting methylation-associated diseases through precision epigenome editing, while identifying critical knowledge gaps in fungal methylome engineering that demand urgent investigation. Full article

Collagen is the main protein found in skin, bone, cartilage, ligaments, tendons and connective tissue, and it can exhibit properties ranging from compliant to rigid or form gradients between these states. The collagen family comprises 28 members, each containing at least one triple-helical domain. These proteins play critical roles in maintaining mechanical characteristics, tissue organization, and structural integrity. Collagens regulate cellular processes such as proliferation, migration, and differentiation through interactions with cell surface receptors. Fibrillar collagens, the most abundant extracellular matrix (ECM) proteins, provide organs and tissues with structural stability and connectivity. In the mammalian myocardial interstitium, types I and III collagens are predominant: collagen I is found in organs, tendons, and bones; collagen II is found in cartilage; collagen III is found in reticular fibers; collagen IV is found in basement membranes; and collagen V is found in nails and hair. Recombinant human collagens, particularly in sponge-like porous formats combined with bone morphogenetic proteins, serve as effective scaffolds for bone repair. Due to their biocompatibility and low immunogenicity, collagens are pivotal in tissue engineering applications for skin, bone, and wound regeneration. Recombinant technology enables the production of triple-helical collagens with amino acid sequences identical to human tissue-derived collagens. This review summarizes recent advances in the molecular functions and recombinant expression of human collagens, with a focus on their biomedical applications. Full article

Mitochondria are involved in a wide array of critical cellular processes from energy production to cell death. The morphology (size and shape) of mitochondrial compartments is highly responsive to both intracellular and extracellular conditions, making these organelles highly dynamic. Nutrient levels and stressors both inside and outside the cell inform the balance of mitochondrial fission and fusion and the recycling of mitochondrial components known as mitophagy. The study of mitochondrial morphology and its implications in human disease and microbial engineering have gained significant attention over the past decade. The yeast Saccharomyces cerevisiae offers a valuable model system for studying mitochondria due to its ability to survive without respiring, its genetic tractability, and the high degree of mitochondrial similarity across eukaryotic species. Here, we review how the interplay between mitochondrial fission, fusion, biogenesis, and mitophagy regulates the dynamic nature of mitochondrial networks in both yeast and mammalian systems with an emphasis on yeast as a model organism. Additionally, we examine the crucial role of inter-organelle interactions, particularly between mitochondria and the endoplasmic reticulum, in regulating mitochondrial dynamics. The dysregulation of any of these processes gives rise to abnormal mitochondrial morphologies, which serve as the distinguishing features of numerous diseases, including Parkinson’s disease, Alzheimer’s disease, and cancer. Notably, yeast models have contributed to revealing the underlying mechanisms driving these human disease states. In addition to furthering our understanding of pathologic processes, aberrant yeast mitochondrial morphologies are of increasing interest to the seemingly distant field of metabolic engineering, following the discovery that compartmentalization of certain biosynthetic pathways within mitochondria can significantly improve chemical production. In this review, we examine the utility of yeast as a model organism to study mitochondrial morphology in both healthy and pathologic states, explore the nascent field of mitochondrial morphology engineering, and discuss the methods available for the quantification and classification of these key mitochondrial morphologies. Full article

Background/Objectives: Determining appropriate cellular objectives is crucial for the system-scale modeling of biological networks for metabolic engineering, cellular reprogramming, and drug discovery applications. The mathematical representation of metabolic objectives can describe how cells manage limited resources to achieve biological goals within mechanistic and environmental constraints. While rapidly proliferating cells like tumors are often assumed to prioritize biomass production, mammalian cell types can exhibit objectives beyond growth, such as supporting tissue functions, developmental processes, and redox homeostasis. Methods: This review addresses the challenge of determining metabolic objectives and trade-offs from multiomics data. Results: Recent advances in single-cell omics, metabolic modeling, and machine/deep learning methods have enabled the inference of cellular objectives at both the transcriptomic and metabolic levels, bridging gene expression patterns with metabolic phenotypes. Conclusions: These in silico models provide insights into how cells adapt to changing environments, drug treatments, and genetic manipulations. We further explore the potential application of incorporating cellular objectives into personalized medicine, drug discovery, tissue engineering, and systems biology. Full article

ABC toxin complexes (Tcs) are tripartite complexes that come together to form nano-syringe-like translocation systems. ABC Tcs are often compared with Bacillus thuringiensis (Bt) toxins, and as such, they have been highly studied as a potential novel pesticide to combat growing insect resistance. Moreover, it is possible to substitute the cytotoxic hypervariable region with alternative peptides, which promise potential use as a novel peptide delivery system. These toxins possess the unique ability to form active chimeric holotoxins across species and display the capability to translocate a variety of payloads across membrane bilayers. Additionally, mutagenesis on the linker region and the receptor binding domains (RBDs) show that mutations do not inherently cause a loss of functionality for translocation. For these reasons, Tcs have emerged as an ideal candidate for targeted protein engineering. However, elucidation of the specific function of each RBD in relation to target receptor recognition currently limits the use of a rational design approach with any ABC Tc. Additionally, there is a distinct lack of targeting and biodistribution data for many Tcs among mammals and mammalian cell lines. Here, we outline two separate strategies for modifying the targeting capabilities of the A subunit (TcA) from Xenorhabdus nematophilus, Xn-XptA2. We identify novel structural differences that make Xn-XptA2 different than other characterized TcAs and display the modular capabilities of substituting RBDs from alternative TcAs into the Xn-XptA2 scaffold. Finally, we show the first, to our knowledge, biodistribution data of any TcA in mice. Full article

Firefly luciferases have been extensively used for bioanalytical applications, including their use as bioluminescent reporters, biosensors, and for bioimaging biological and pathological processes. Due to their intrinsic pH- sensitivity, in recent years we have demonstrated that firefly luciferases can also be harnessed as color- tuning sensors of intracellular pH. However, it is known that mammalian cells require temperatures higher than 36 °C, which red-shift the bioluminescence spectra of most firefly luciferases, decreasing their activities and the resolution of ratiometric pH analysis. Therefore, we prospected and engineered novel pH-sensitive firefly luciferases for mammalian cells. We humanized the luciferases of Amydetes vivianii (Amy-Luc) and Cratomorphus distinctus (Crt-Luc) fireflies, inserted them into the pCDNA3 vector, and compared their bioluminescence and pH-sensing properties with those of Macrolampis firefly luciferase (Mac-Luc) inside fibroblasts. The transfected COS-1 with Mac-Luc and Crt-Luc displayed lower bioluminescence activity and considerably red-shifted spectra (611 and 564 nm, respectively) at 37 °C, whereas Amy-Luc displayed the highest bioluminescence activity and spectral stability at 37 °C inside cells, displaying the most blue-shifted spectrum at such temperatures (548 nm) and the best spectral resolution at different pH values, making it possible to ratiometrically estimate the pH from 6.0 to 8.0. These results show that Amy-Luc is a novel brighter reporter gene and suitable pH- indicator for mammalian cells. Furthermore, whereas at pH 8.0 the spectrum was thermally stable, at pH 6.0 Amy-Luc showed higher temperature sensitivity, raising the possibility of using this luciferase as an intracellular temperature sensor. Thus, the improved bioluminescence properties as compared to existing luciferases could offer advantages for in vivo imaging and pH- sensing for the study of mammalian cellular physiology. Full article

Electrospinning can be used to prepare nanofiber mats from diverse polymers and polymer blends. A large area of research is the application of nanofibrous membranes for tissue engineering. Typically, cell adhesion and proliferation as well as the viability of mammalian cells are tested by seeding the cells on substrates, cultivating them for a defined time and finally dyeing them to enable differentiation between cells and substrates under a white light or fluorescence microscope. While this procedure works well for cells cultivated in well plates or petri dishes, other substrates may undesirably also be colored by the dye. Here we show investigations of the optical contrast between dyed CHO DP-12 (Chinese hamster ovary) cells and different electrospun nanofiber mats, dyed with haematoxylin-eosin (H&E), PromoFluor 488 premium, 4,6-diamidino-2-phenylindole (DAPI) or Hoechst 33342, and give the optimum dyeing parameters for maximum optical contrast between cells and nanofibrous substrates. Full article

Abnormal heart valve function is a major cause of heart disease and is often associated with high rates of morbidity and mortality from heart disease. Focusing on the field of valve replacement, tissue-engineered heart valves are becoming popular and attracting more attention. In this work, an artificial heart valve scaffold based on polycaprolactone/polyurethane (PCL/PU) three-layer composite fibers was prepared by 3D printing and electrospinning, including an inner PCL layer, a middle PCL/PU layer, and an outer PCL layer. The obtained valve scaffold had strong circumferential mechanical properties when PCL/PU = 1:2, and its elastic modulus was 14.7 MPa, similar to that of natural valve. The heart valve scaffold had good cytocompatibility, and the final cell survival rate was 99.8% after 14 days of cell culture. The layered structure makes the artificial heart valve more similar to the natural structure of the mammalian heart, which is conducive to cell proliferation and differentiation, and provides a reference solution for interventional treatment of heart valves. Full article

This review is devoted to experimental studies and modeling in the field of mechanical and physical properties of polymer concretes and polymer-modified concretes. The review analyzes studies carried out over the past two years. The paper examines the properties of polymer concretes based on various polymer resins and presents the advantages and disadvantages of various models developed to predict the mechanical properties of materials. Based on data in the literature, the most promising polymers for use in the field of road surface repair are polymer concretes with poly(meth)acrylic resins. It was found that the most adequate and productive models are the deep machine learning model—using several hidden layers that perform calculations based on input parameters—and the extreme gradient boosting model. In particular, the extreme gradient boosting model showed high R² values in forecasting (in the range of 0.916–0.981) when predicting damping coefficient and ultimate compressive strength. In turn, among the additives to Portland cement concrete, the most promising are natural polymers, such as mammalian gelatin and cold fish gelatin, and superabsorbent polymers. These additives allow for an improvement in compressive strength of 200% or more. The review may be of interest to engineers specializing in building construction, materials scientists involved in the development and implementation of new materials into production, as well as researchers in the interdisciplinary fields of chemistry and technology. Full article

Lignin, the most abundant renewable aromatic polymer, has been shown to suppress the growth of mammalian tumor cells. Despite extensive studies on lignin structure and its engineering, there is little information on the biological activity of lignin in relation to its molecular structure or the molecular mechanisms by which lignin suppresses tumor cells in mammalian species. Here, we prepared microwave-assisted acid-catalyzed solvolysis lignin (MASL) from Japanese cedar and Eucalyptus globulus and assessed its effects on human and mouse tumor cells. SEC indicated that MASL consists of oligomeric aromatics from the woody plants. Our data showed that MASL significantly reduced the viability of tumor cells by modulating apoptotic pathways. MASL treatment upregulated TNF-α, Fas, and FasL expression levels, while suppressing anti-apoptotic NF-κB and mTOR pathways in tumor cells. In vivo experiments were also performed using tumor-bearing mice to confirm the anti-tumor effects of MASL. Repetitive administrations of a MASL (YM CL1T) significantly inhibited tumor growth in mice in association with elevation of caspase 3 expression. These findings strongly suggest the potential usefulness of low-molecular-weight lignin as an effective therapeutic against malignancies. Full article