Biotechnological Applications Research Articles

Agarose Degrading Potential and Whole Genome Sequence Analysis of Marine Bacterium Aliagarivorans sp. Strain DM1 Isolated from the Arabian Sea.

In recent years, agar-degrading bacteria have gained significant interest due to their biotechnological, environmental, microbiological, and industrial applications. Agar poses challenges such as marine waste accumulation, difficult industrial processing, limited natural degradability, and sustainability concerns due to high demand and overharvesting of red algae. The present study addresses the need for efficient agar-degrading microorganisms by isolating Aliagarivorans sp. strain DM1 from biofilm on fabric surfaces in the intertidal regions of the Arabian Sea, India. Phylogenetic analysis revealed that strain DM1 is closely related to Aliagarivorans taiwanensis AAT1T, and it exhibited significant agar-degrading activity on Zobell marine agar plates. Whole genome sequencing of Aliagarivorans sp. strain DM1, conducted using the Illumina NovaSeq platform, yielded a genome size of 4,898,415bp with an average G + C content of 53.3%. The genome includes 4,518 predicted protein-coding genes (CDS), 86 transfer RNA (tRNA) genes, and two ribosomal RNA (rRNA) genes, with thirteen predicted agarases identified. The highest enzyme activity recorded was 51.00 U mL-1 on the 6th day of incubation using 10% inoculum, with optimal conditions of pH 8-9, 0.8M NaCl, and temperatures between 50 and 60°C. These findings underscore the promise of Aliagarivorans sp. strain DM1 in developing efficient enzymatic processes that can be applied in various biotechnological and industrial fields, including waste management and agaro-oligosaccharide production. Furthermore, strain DM1 possesses several key characteristics that enhance its adaptability and utility in marine and industrial applications, surpassing closely related strains in enzyme stability, environmental tolerance, and industrial versatility.

Evaluation of the Robustness Under Alkanol Stress and Adaptability of Members of the New Genus Halopseudomonas

Many species of the genus Pseudomonas are known to be highly tolerant to solvents and other environmental stressors. Based on phylogenomic and comparative genomic analyses, several Pseudomonas species were recently transferred to a new genus named Halopseudomonas. Because of their unique enzymatic machinery, these strains are being discussed as novel biocatalysts in biotechnology. In order to test their growth parameters and stress tolerance, five Halopseudomonas strains were assessed regarding their tolerance toward different n-alkanols (1-butanol, 1-hexanol, 1-octanol, 1-decanol), as well as to salt stress and elevated temperatures. The toxicity of the solvents was investigated by their effects on bacterial growth rates and presented as EC50 concentrations. Hereby, all Halopseudomonas strains showed EC50 values up to two-fold lower than those previously detected for Pseudomonas putida. In addition, the activity of the cis-trans isomerase of unsaturated fatty acids (Cti), which is an urgent stress response mechanism known to be present in all Pseudomonas species, was monitored in the five Halopseudomonas strains. Although several of the tested species were known to contain the cti gene, no significant phenotypic activity could be detected in the presence of the assayed stressors. A bioinformatic analysis of eight cti-carrying Halopseudomonas strains examining promotor binding sites, binding motifs and signal peptides showed that most of the cti genes have a lipoprotein signal peptide and promotor regions and binding motifs that do not coincide with those of Pseudomonas. These insights represent putative reasons for the absence of the expected Cti activity in Halopseudomonas, which in turn has always been observed in cti-carrying Pseudomonas. The lack of Cti activity under membrane stress conditions when the cti gene is present has never been documented, and this could represent potential negative implications on the utility of the genus Halopseudomonas for some biotechnological applications.

Highly Efficient Red/Near‐Infrared Phosphorescence from Doped Crystals

AbstractOrganic red/near‐infrared (NIR) room temperature phosphorescence (RTP) materials with low toxicity and facile synthesis are highly sought after, particularly for applications in biotechnology and encryption. However, achieving efficient red/NIR RTP emitters has been challenging due to the weak spin‐orbit coupling of organics and the rapid nonradiative decay imposed by the energy gap law. Here we demonstrate highly efficient red/NIR RTP with boosted quantum yields (Φps) of up to 32.96 % through doping the thionated derivatives of phthalimide (PAI) (MTPAI and DTPAI) into PAI crystals. The red‐shifted photoluminescence (PL) stems from a combination of the external heavy atom effect and the formation of emissive clusters centered around electron‐rich sulfur atoms. Furthermore, the dopants enhance exciton generation efficiency and facilitate energy transfer from smaller PAI units to larger aggregates, leading to dramatically increased Φp. This strategy proves universal, opening possibilities for acquiring long‐wavelength RTP with tunable photophysical properties. The doped crystals exhibit promising applications in optical waveguides and encryption paper/ink. This research provides a practical approach to obtaining long‐wavelength RTP materials and offers valuable insights into the mechanisms governing host‐guest systems.

Highly Efficient Red/Near-Infrared Phosphorescence from Doped Crystals.

Organic red/near-infrared (NIR) room temperature phosphorescence (RTP) materials with low toxicity and facile synthesis are highly sought after, particularly for applications in biotechnology and encryption. However, achieving efficient red/NIR RTP emitters has been challenging due to the weak spin-orbit coupling of organics and the rapid nonradiative decay imposed by the energy gap law. Here we demonstrate highly efficient red/NIR RTP with boosted quantum yields (Φps) of up to 32.96 % through doping the thionated derivatives of phthalimide (PAI) (MTPAI and DTPAI) into PAI crystals. The red-shifted photoluminescence (PL) stems from a combination of the external heavy atom effect and the formation of emissive clusters centered around electron-rich sulfur atoms. Furthermore, the dopants enhance exciton generation efficiency and facilitate energy transfer from smaller PAI units to larger aggregates, leading to dramatically increased Φp. This strategy proves universal, opening possibilities for acquiring long-wavelength RTP with tunable photophysical properties. The doped crystals exhibit promising applications in optical waveguides and encryption paper/ink. This research provides a practical approach to obtaining long-wavelength RTP materials and offers valuable insights into the mechanisms governing host-guest systems.

Review of Mimusops zeyheri Sond. (Milkwood): Distribution, Utilisation, Ecology and Population Genetics.

Mimusops zeyheri Sond. (Milkwood) is an indigenous fruit tree species with considerable ecological, cultural, and nutritional significance that remains underexploited. This review synthesizes current knowledge on its distribution, taxonomy, phytochemistry, ethnomedicinal applications, ecological functions, genetic diversity, and biotechnological potential. A systematic literature search, spanning 1949 to April 2024, yielded 87 relevant publications from an initial 155. Mimusops zeyheri plays a crucial role in supporting the cultural traditions and economic activities of Indigenous Southern African Communities. Its distribution encompasses South, East, and Southern Tropical Africa, with substantial populations across South African provinces. Ethnomedicinally, various plant parts treat conditions including wounds, gastrointestinal issues, and diabetes. The leaves (34%) and roots (32%) are used, with infusion (33%) and decoction (31%) as primary preparation methods. Oral administration (70%) is the most common, primarily addressing skin conditions (18%). Despite its nutritional richness, a standardized nutrient profile is lacking. Limited genetic diversity studies underscore the need for further research. This study highlights Mimusops zeyheri's multifaceted importance and research gaps, particularly in other Southern African countries. Future investigations should focus on comprehensive phytochemical analysis, ethnomedicinal validation, ecological conservation, genetic diversity assessment, and biotechnological applications. Multidisciplinary collaborations are recommended to promote sustainable utilization while preserving traditional practices.

Whole genome sequencing of a Domibacillus strain from the Cuatro Ciénegas basin that tends to act as an altruist.

Domibacillus sp. 8LH is a whitish bacterium isolated from the pools of the Cuatro Cienegas Basin (CCB) in the state of Coahuila and belongs to the Bacillaceae family. It grows in circular colonies of about 6 mm in diameter and is capable of forming biofilms. This strain was identified because, in previous experiments in our laboratory, it presented altruistic interactions when co-cultured with bacteria of the genus Bacillus that participate in the nitrogen cycle. This altruistic behavior confers to this Domibacillus strain a potential use for the construction of bacterial consortia with diverse biotechnological applications.

Structures of a DNA Polymerase Caught while Incorporating Responsive Dual-Functional Nucleotide Probes.

Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5-heterocycle-modified environment-sensitive 2'-deoxyuridine-5'-triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual-app probes, combining a fluorophore with X-ray anomalous scattering Se or 19F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, FBFdUTP's responsiveness enabled real-time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next-generation nucleotide probe development.

Structures of a DNA Polymerase Caught while Incorporating Responsive Dual‐Functional Nucleotide Probes

Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5‐heterocycle‐modified environment‐sensitive 2′‐deoxyuridine‐5′‐triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual‐app probes, combining a fluorophore with X‐ray anomalous scattering Se or 19F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, FBFdUTP's responsiveness enabled real‐time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next‐generation nucleotide probe development.

Manipulation of Cancer Cell Spheroids by Photovoltaic Tweezers: Determination of Their Charge State

Photovoltaic optoelectronic tweezers (PVOT), based on the bulk photovoltaic effect presented by certain ferroelectric crystals, have emerged as a versatile tool to manipulate and/or trap micro‐ and nano‐objects. Very recently, an ingenious method has enabled to extend PVOT manipulation to aqueous solution droplets opening the door to applications in optofluidics and biotechnology. The photovoltaic crystal is placed on the bottom of a cuvette filled with paraffin oil while the droplet is hanging from the air–oil interphase where it is manipulated. Herein, this method is used to investigate the feasibility of manipulating hybrid droplets as carriers containing a cancer multicellular spheroid inside cell culture medium. Droplets of two cancer cell lines, MCF‐7 and U‐87 MG, have been successfully manipulated by light‐induced electrical fields generated in the photovoltaic platform. From the droplet dynamics, which includes attractive and repulsive behaviors from the illuminated crystal region, the charge state of the cell culture medium and the spheroid are investigated. The results show that they are positively and negatively charged, respectively. Moreover, analyzing the migration kinetics of water, cell culture medium, and hybrid droplets, to the light of a proposed theoretical model, the charge density of both cell line spheroids has been estimated.

A survey of the Desulfuromonadia “cytochromome” provides a glimpse of the unexplored diversity of multiheme cytochromes in nature

BackgroundMultiheme cytochromes c (MHC) provide prokaryotes with a broad metabolic versatility that contributes to their role in the biogeochemical cycling of the elements and in energy production in bioelectrochemical systems. However, MHC have only been isolated and studied in detail from a limited number of species. Among these, Desulfuromonadia spp. are particularly MHC-rich. To obtain a broad view of the diversity of MHC, we employed bioinformatic tools to study the cytochromome encoded in the genomes of the Desulfuromonadia class.ResultsWe found that the distribution of the MHC families follows a different pattern between the two orders of the Desulfuromonadia class and that there is great diversity in the number of heme-binding motifs in MHC. However, the vast majority of MHC have up to 12 heme-binding motifs. MHC predicted to be extracellular are the least conserved and show high diversity, whereas inner membrane MHC are well conserved and show lower diversity. Although the most prevalent MHC have homologues already characterized, nearly half of the MHC families in the Desulforomonadia class have no known characterized homologues. AlphaFold2 was employed to predict their 3D structures. This provides an atlas of novel MHC, including examples with high beta-sheet content and nanowire MHC with unprecedented high numbers of putative heme cofactors per polypeptide.ConclusionsThis work illuminates for the first time the universe of experimentally uncharacterized cytochromes that are likely to contribute to the metabolic versatility and to the fitness of Desulfuromonadia in diverse environmental conditions and to drive biotechnological applications of these organisms.

Microalgae to remove pharmaceutical and personal care products (PPCPs) from wastewater

Substances used for medicinal, cosmetic, hygiene, and health care objectives are included in the category of pharmaceuticals and personal care products (PPCPs). They are a major source of public concern because of their ubiquitous manufacturing, usage, and careless disposal of expired products into the environment. Many PPCPs, including antibiotics, analgesics, endocrine disruptors, and microbial/disinfecting agents, are commonly detected in freshwater systems, groundwater, and wastewater treatment effluents in amounts ranging from nanograms per liter to milligrams per milliliter. Additionally, these compounds frequently show persistence and accumulate in biological tissue, eventually finding their way into crops, vegetables, and drinking water supplies. Because many PPCPs are known to have the ability to upset ecosystems and/or provide health hazards, they are categorized as “emerging contaminants.” The research on the occurrence, fate, and possible health and environmental hazards related to PPCPs in both aquatic and terrestrial habitats is thoroughly reviewed in this work. It also covers reported cases of danger or health concerns in humans, although a full assessment may not be possible given the limitations of the data at hand. This review aims to provide a comprehensive and well-focused overview of the current understanding of bioremediation strategies for the removal of pharmaceuticals and personal care products (PPCPs), with an emphasis on the application of macroalgae, microalgae, and aquatic macrophytes. It examines the physicochemical properties of PPCPs and their potential risks to environmental and human health. Additionally, the review explores the potential and challenges associated with the broader application of biotechnologies employing algae and aquatic macrophytes. This includes research efforts to correlate the operational parameters of these biotechnologies with the primary mechanisms responsible for PPCP removal. In conclusion, algae and macrophytes present promising, eco-friendly solutions for wastewater treatment, significantly contributing to the mitigation of PPCP contamination.

Physicochemical and thermodynamic properties of purified rhodanese from A. welwitschiae LOT1 and the cyanide detoxification potential of the enzyme.

Rhodanese, the primary cyanide-detoxifying enzyme, plays a crucial role in mitigating the harmful effects of cyanide present in various industrial waste materials, such as battery manufacturing effluents. The bioremediation of cyanide-contaminated environments relies on efficient detoxification mechanisms, making rhodanese a valuable enzyme for biotechnological applications. This research aimed to investigate the biochemical properties of purified rhodanese produced by Aspergillus welwitschiae LOT1, a fungal strain with promising cyanide detoxification capabilities. The purified rhodanese was obtained through fermentation, precipitation, and chromatographic separations, resulting in a homogeneous band of approximately 58kDa with a specific activity of 374 RU/mg, 28-fold purification, and 14% recovery. The enzyme exhibited optimal cyanide detoxification at pH 7 and 60°C, with stability observed between 30 and 50°C and pH 8-10. All metal ions examined except for Cu2+ enhanced the cyanide-degrading ability of rhodanese. Notably, the enzyme demonstrated a high substrate preference for Na2S2O3 and followed a first-order kinetic model and free energy, ΔG of 61.3kJ/mol, making it a promising candidate for biotechnological applications. Overall, this study provides valuable insights into the biochemical properties of rhodanese from A. welwitschiae LOT1, highlighting its potential for efficient cyanide detoxification and bioremediation.

Discovery and Biosynthesis of Sulfenicin and Its New-to-Nature Acylsulfenic Acid Functional Group.

Life's organic molecules are built with diverse functional groups that enable biology by fine tuning intimate connections through time and space. As such, the discovery of new-to-nature functional groups can expand our understanding of the natural world and motivate new applications in biotechnology and biomedicine. Herein we report the genome-aided discovery of sulfenicin, a novel polyketide-nonribosomal peptide hybrid natural product from a marine Streptomyces bacterium bearing a unique acylsulfenic acid functionality. Through a series of heterologous biosynthesis, functional genetics, and enzymatic reconstitution experiments, we show that this previously described synthetic functional group is biologically assembled by a set of enzymes from both primary and secondary metabolism, including a novel flavin-dependent S -hydroxylase that hydroxylates a thiocarboxylic acid's sulfur atom. While the sulfenicin biosynthetic gene cluster is presently without parallel in public databases, acylsulfenic acid-encoding enzymes are widely distributed in bacterial genomes, implying that this labile functional group may similarly have a broad distribution among specialized metabolites.

Optimized Rhamnolipid Production by a Pseudomonas marginalis C9 Strain Isolated from a Biopurification System to Enhance Pesticide Solubilization

Biopurification systems designed for pesticide treatment are a source of diverse bacteria with environmental and biotechnological applications, including Pseudomonas marginalis C9, which has been reported as a biosurfactant-producing bacterium. The optimization of biosurfactant produced from P. marginalis C9 to enhance the solubility of a hydrophobic pesticide of environmental interest was investigated. The response surface methodology (RSM) was used to optimize the combined effect of the initial pH (5–9), agitation (100–300 rpm), and temperature (24–32 °C) on biosurfactant production. A DASbox® automated mini-bioreactor system was used to evaluate the critical factors in biosurfactant production using a full factorial design (FFD). The results showed that the optimal culture conditions using RSM were a pH of 8.5, a temperature of 25 °C, and agitation at 200 rpm. The extraction yield of the biosurfactant was 7.40 g L−1, the surface tension was reduced to 27.45 mN m−1, and the critical micelle concentration (CMC) was 48.9 mg L−1. The FFD analysis indicated that a high agitation rate (300 rpm) strongly influenced the biosurfactant activity, regardless of the inlet oxygen supply (0.5–1.5 vvm). The rhamnolipid increased the water solubility of chlorpyrifos by 11.2- and 21.7-fold at the CMC and twice the CMC, respectively.

The signal peptide of BmNPV GP64 activates the ERAD pathway to regulate heterogeneous secretory protein expression

As a powerful eukaryotic expression vector, the baculovirus expression vector system (BEVS) is widely applied to the production of heterogeneous proteins for research and pharmaceutical purposes, while optimization of BEVS remains a work in progress for membrane or secreted protein expression. In this study, the impact of the signal peptide (SP) derived from Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 protein on protein expression, secretion, and the endoplasmic reticulum-associated degradation (ERAD) pathway were investigated in BmN cells and BEVS. Transient expression studies in BmN cells revealed that SP alters the localization and expression levels of recombinant proteins, reducing intracellular accumulation while enhancing secretion efficiency. Quantitative analysis demonstrated that SP-mediated secretion was markedly higher compared to controls, albeit with lower total expression levels. Further exploration into SP-mediated ERAD pathway activation showed increased expression of BiP and other ERAD-associated genes (PDI, UFD1, S1P, and ASK1), correlating with higher SP-driven protein expression levels. RNA interference (RNAi) experiments elucidated that knockdown of ERAD-associated genes enhances both the secretion efficiency of SP-guided proteins and the infectivity of BmNPV. Particularly, interference with BiP demonstrated the most pronounced effect on protein secretion enhancement. Viral infection experiments further supported these findings, showing upregulated ERAD-associated genes during BmNPV infection, indicating their role in viral protein processing and infectivity. In conclusion, this study elucidates the complex interplay between SP-mediated protein secretion, ERAD pathway activation, and viral infectivity in BmNPV-infected cells. These insights suggest strategies for optimizing recombinant protein production and viral protein processing in baculovirus expression systems, with potential implications for biotechnological and biomedical applications. Further research could refine our understanding and manipulation of protein secretion pathways in insect cell-based expression systems.

Ramachandran-like Conformational Space for DNA.

DNA's ability to exist in a wide variety of structural forms, subforms, and secondary motifs is fundamental to numerous biological processes and has driven the development of biotechnological applications. Major determinants of DNA flexibility are the multiple torsional degrees of freedom around the phosphodiester backbone. This high complexity can be rationalized by using two pseudotorsional angles linking atoms P and C4', from which Ramachandran-like plots can be built. In this contribution, we explore the distribution of η (eta: C4'i-1-Pi-C4'i-Pi+1) and θ (theta: Pi-C4'i-Pi+1-C4'i+1) angles in known experimental structures retrieved from the Protein Data Bank (PDB), subdividing the conformational space into different datasets. After the removal of the canonical/helical conformations typical of the B-form, we find the existence of a conformational map with clearly permitted and forbidden regions. Some of these regions are populated with specific DNA forms, like Z- or A-DNA, or by specific secondary motifs, like G-quadruplexes and junctions. We evaluated the sequence dependency and energy relationship among the high-density regions identified in the η-θ space. Furthermore, we analyzed the effect produced by proteins and cations when bound to DNA, finding that specific proteins produce some nonhelical conformations, while other regions appear to be stabilized by divalent cations.

Effects of poultry by-product meal and complete replacement of fish oil with alternative oils on growth performance and gut health of rainbow trout (Oncorhynchus mykiss): a FEEDNETICS™ validation study

BackgroundAquaculture, traditionally a form of biotechnology, has evolved to integrate innovative biotechnological applications, such as advanced feed formulations, aimed at improving the growth performance and health of farmed fish species. In the present study, the effects of feeding rainbow trout with novel feed formulations were investigated. Fish growth, gut and liver morphology, the concentration of fatty acids in the fillet, and volatile fatty acids in the gut were assessed. The study also validated scenarios from in vivo experiments using a nutrient-based model called FEEDNETICS™. This globally used model serves as a tool for data interpretation and decision support in the context of precision fish farming.MethodsAlternative protein and oil sources, including poultry by-product meal (PBM) and natural algae oil, were explored as sustainable replacements for fishmeal (FM) and fish oil (FO). A 90-day feeding trial was conducted using rainbow trout, comparing two isoproteic, isolipidic and isoenergetic diets. The control diet contained 15% FM, 5% PBM, and 8% FO, while the test diet replaced FM with 15% PBM and 5% feather meal hydrolysate (FMH), and fully substituted FO with VeraMaris® natural algae oil and rapeseed oil.ResultsPBM successfully replaced FM protein without negatively affecting feed intake, growth performance or feed utilization in trout. The combination of PBM and natural algae oil was well tolerated by the trout and showed no negative effects on gut health. A detailed analysis of fatty acids in the fillet revealed that PUFAs of the n3 and n6 series were significantly higher in the PBM group than in the FM group. Values of fatty acid-related health indexes, including atherogenicity index, and thrombogenicity index, confirmed the high nutritional value of trout filet, thus representing a healthy product for human. In addition, the predictions using the FEEDNETICS™ indicated that the tested novel alternative formulations are economically viable. The validation of the model for fish growth resulted in a Mean Absolute Percentage Error (MAPE) of 8%.ConclusionsThe FEEDNETICS™ application enhances our ability to optimize feeding strategies and improve production efficiency in the aquaculture industry. VeraMaris® algae oil and PBM could serve as viable and sustainable raw materials for fish feed, promoting environmentally friendly aquaculture practices.

Uncovering the roles of the scaffolding protein CsoS2 in mediating the assembly and shape of the α-carboxysome shell.

Carboxysomes are a paradigm of organelle-like structures in cyanobacteria and many proteobacteria. These nanoscale compartments enclose Rubisco and carbonic anhydrase within an icosahedral virus-like shell to improve CO2 fixation, playing a vital role in the global carbon cycle. Understanding how the carboxysomes are formed is not only important for basic research studies but also holds promise for repurposing carboxysomes in bioengineering applications. In this study, we focuses on a specific scaffolding protein called CsoS2, which is involved in facilitating the assembly of α-type carboxysomes. By deciphering the functions of different parts of CsoS2, especially its middle region, we provide new insights into how CsoS2 drives the stepwise assembly of the carboxysome at the molecular level. This knowledge will guide the rational design and reprogramming of carboxysome nanostructures for many biotechnological applications.

Assessment of a Structurally Modified Alternanthera Mosaic Plant Virus as a Delivery System for Sarcoma Cells

The virions of plant viruses and their structurally modified particles (SP) represent valuable platforms for recombinant vaccine epitopes and antitumor agents. The possibility of modifying their surface with biological compounds makes them a tool for developing medical biotechnology applications. Here, we applied a new type of SP derived from virions and virus-like particles (VLP) of Alternanthera mosaic virus (AltMV) and well-studied SP from Tobacco mosaic virus (TMV). We have tested the ability of SP from AltMV (AltMV SPV) and TMV virions also as AltMV VLP to bind to and penetrate Ewing sarcoma cells. The adsorption properties of AltMV SPV and TMV SP are greater than those of the SP from AltMV VLP. Compared to normal cells, AltMV SPV adsorbed more effectively on patient-derived sarcoma cells, whereas TMV SP were more effective on the established sarcoma cells. The AltMV SPV and TMV SP were captured by all sarcoma cell lines. In the established Ewing sarcoma cell line, the effectiveness of AltMV SPV penetration was greater than that of TMV SP. The usage of structurally modified plant virus particles as a platform for drugs and delivery systems has significant potential in the development of anticancer agents.

Light-guided actin polymerization drives directed motility in protocells.

Motility is a hallmark of life's dynamic processes, enabling cells to actively chase prey, repair wounds, and shape organs. Recreating these intricate behaviors using well-defined molecules remains a major challenge at the intersection of biology, physics, and molecular engineering. Although the polymerization force of the actin cytoskeleton is characterized as a primary driver of cell motility, recapitulating this process in protocellular systems has proven elusive. The difficulty lies in the daunting task of distilling key components from motile cells and integrating them into model membranes in a physiologically relevant manner. To address this, we developed a method to optically control actin polymerization with high spatiotemporal precision within cell-mimetic lipid vesicles known as giant unilamellar vesicles (GUVs). Within these active protocells, the reorganization of actin networks triggered outward membrane extensions as well as the unidirectional movement of GUVs at speeds of up to 0.43 µm/min, comparable to typical adherent mammalian cells. Notably, our findings reveal a synergistic interplay between branched and linear actin forms in promoting membrane protrusions, highlighting the cooperative nature of these cytoskeletal elements. This approach offers a powerful platform for unraveling the intricacies of cell migration, designing synthetic cells with active morphodynamics, and advancing bioengineering applications, such as self-propelled delivery systems and autonomous tissue-like materials.