Abstract: Human neutrophil elastase (HNE) is a central mediator of neutrophil-driven inflammation. Yet, despite decades of research and drug development, therapies targeting HNE have not consistently translated into clear clinical benefits. We suggest that this translational gap partly arises from how HNE has traditionally been conceptualized, as a single enzyme to inhibit. In biological systems, however, HNE operates within a complex and tightly regulated network of proteases and inflammatory mediators. This network is spatially compartmentalized and strongly in-fluenced by local redox conditions, making HNE activity highly context dependent. From a systems perspective, HNE acts as an amplifier of inflammation. Its extracellular activity connects several pathological processes, including activation of innate immunity, extracellular matrix degradation, disruption of epithelial and endothelial barriers, and the transition toward chronic inflammation. In this review, we integrate insights from enzymology, systems biology, and clinical research to reassess the development of HNE inhibitors, ranging from endogenous anti-proteases to more recent reversible synthetic compounds. Despite their chemical and pharma-cological diversity, many of these strategies have encountered similar limitations. We therefore argue that future therapeutic approaches should move beyond the inhibition of HNE as an iso-lated target and instead aim to modulate the broader protease network, with particular attention to drug–target kinetics and precise delivery to disease-relevant microenvironments.

Abstract: The ability to precisely edit genetic characteristics with a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) immunity complex is a revolutionary advance in science. Originally discovered in bacteria as part of a natural defense mechanism against viruses, CRISPR/Cas provides a precise, efficient, and relatively simple method for editing genes in microbes, plants, animals, and humans. The process relies on the Cas protein, an enzyme that cleaves and unwinds DNA at targeted locations. This process is guided by RNA sequences complementary to the DNA or RNA sequence of interest, allowing for changes to the genome through innate non-homologous end joining (NHEJ) and homology-directed repair (HDR). The potential applications of CRISPR/Cas are immense and in agriculture, is facilitating crop development with resistance to abiotic, biotic, and agronomic characteristics that improve yield, quality, and food security. Gene editing also facilitates the relatively rapid modification of regulatory and complex pathways that enable studies to advance our understanding of gene function. This review provides an update of the fast-evolving CRISPR/Cas modification of important crops to address emerging global population, environmental and climate challenges.

Abstract:

The alkaline pectate lyase A from Paenibacillus barcinonensis, encoded by pelA (GenBank accession no. CAB40884), is an enzyme with high activity on pectin and potential application in sustainable industrial biotechnology. In this study, pelA was expressed in Saccharomyces cerevisiae by using different domains of the cell wall protein Pir4 as translational fusion partners. Given the presence of five potential N-glycosylation sites in the amino acid sequence coded by pelA, two of them in conserved regions of class III pectate lyases, the effect of glycosylation on the enzymatic activity of the recombinant enzyme was investigated by expressing the recombinant fusion proteins in both, standard and glycosylation deficient strains of S. cerevisiae. Correct targeting of the recombinant fusion proteins was confirmed by Western blot analysis using Pir-specific antibodies, whilst enzymatic activity on polygalacturonic acid was demonstrated on both plate assays and colorimetric assays. Hyper- glycosylation of the enzyme when expressed in the standard strain of S. cerevisiae did not occur, however maximum activities were over two and a half times higher when the enzyme was expressed in the glycosylation deficient strain, suggesting a better adaptation of this strain to the secretion of the functional enzyme. Notably, pectate lyase activity was approximately fourfold higher when the pelA gene was expressed in this yeast strain compared to its expression in a prokaryotic host such as Bacillus subtilis or Escherichia coli.

Abstract: The problem of aligning humans and artificial intelligences can be understood in terms of minimizing externalities between them. However, economics cannot define externality because it contradicts the rationality assumption. This paper applies the homeostatic principles, from anatomical homeostasis to its disorder – cancer, to define externality. Drawing upon the perspective of cancer as a problem of scaling cellular collectives, this paper shows how to redefine both externality and rationality in terms of cognitive light cones (which demarcate the scale of goals any agent can pursue). We propose that cognitive light cones are constructed out of interoceptive signals for the purpose of anatomical homeostasis. We show that externalities can be understood in terms of anatomical homeostasis and derive some important implications for AI alignment, including the possibility of using market mechanisms enable the mutual co-construction of alignment between artificial intelligences and humans.

Abstract: Fucoidan is a sulfated polysaccharide derived from brown seaweed, reported to possess diverse biological activities that make it a molecule of great interest for nutraceutical and biomedical applications. A significant challenge to its wider use is the limited understanding of the relationship between fucoidan’s structural and chemical characteristics with biological activity. This gap was addressed by detailed chemical characterization of commercial fucoidan extracts and biological activity testing (antioxidant, antiviral, antifungal, antibacterial and prebiotic) to elucidate potential relationships for further study. Fucoidan extracts exhibited multiple bioactivities, notably antioxidant activity, antiviral activity against Nipah virus, antifungal activity against Candida dubliensis and prebiotic effects on Lactobacillus casei. No antifungal activity against Candida albicans, Candida auris and Cryptococcus neoformans, nor antibacterial effects against Klebsiella pneumoniae, were observed. A correlation analysis to identify potential key quality attributes of fucoidan was employed, suggesting that high fucose content may be important for antioxidant, antiviral, antifungal, and prebiotic activities. A high sulfation degree was also identified as potentially optimal for these bioactivities. This work also addressed a debate in the literature regarding the optimal molecular weight for bioactivity, demonstrating that this likely depends on the specific microbe to which a fucoidan extract is applied, with smaller molecular weights required for antioxidant and antiviral bioactivities and larger molecular weights for antifungal and prebiotic activities. This study demonstrates that a formalized comparative approach linking chemical and structural data with results from biological activity assessments can effectively identify important fucoidan characteristics underpinning specific bioactivity. The results of which are worthy of further validation.

Abstract: Freeze-drying of Platelet-rich plasma and its effect on wound healing: An in vivo pilot case study. Himanshu Bansal1,2*, Alnkrita Bansal1,2, Irfan Khan2, Anupama Bansal1, Shahnawaz Hussein Khan3, Jerry Leon4, Mustafa al Maini5, Matias Fernandez Viña6 1Mother Cell Spinal Injury and Stem Cell Research, Anupam Hospital, Rudrapur, Uttarakhand, India 2Revita Life Sciences, Rudrapur, Uttarakhand, India 3Department of regenerative medicine, Lovinium Biotech, Asturias, Spain 4PMR Advance Health Institute Mayaguez, Puerto Rico, USA 5Mafraq Hospital, Sheikh Shakhbout Medical City, Abu Dhabi, United Arab Emirates 6Consultant, Stem Cell Therapy, Argentina *Corresponding author Dr. Himanshu Bansal, MD, Consultant Regenerative Medicine, Revita Lifesciences, Rudrapur, India-263153 Phone: 09634501234 Email: hbansal@drhbf.org   Abstract Background: Platelet-rich plasma (PRP) is widely used in regenerative medicine because of its high content of growth factors and cytokines that promote tissue repair. However, fresh PRP is limited by the rapid loss of bioactivity, compositional variability, and storage challenges. Freeze-dried PRP (FD-PRP) is a promising alternative with potential for long-term storage and standardized therapeutic efficacy. Objective: This study aimed to evaluate the biological activity, growth factor preservation, and wound healing efficacy of FD-PRP compared with fresh PRP using in vitro and in vivo assays. Methods: PRP from 20 healthy donors was standardized to 5 billion platelets per 4 mL and freeze-dried using proprietary stabilizers. Platelet morphology, aggregation, and growth factor concentrations (PDGF, TGF-β1, VEGF, EGF, bFGF, and IGF-1) were assessed before and after lyophilization. The stability of the samples was monitored over 26 weeks. In vitro wound healing was assessed in human mesenchymal stem cells (MSCs) using scratch assays. A pilot in vivo study evaluated the efficacy of FD-PRP in accelerating wound closure in 10 patients over three weeks. Results: FD-PRP retained 82.05% of platelets post-lyophilization, with preserved morphology and aggregation. Growth factor levels were maintained or increased in FD-PRP, notably TGF-β1 (2.1-fold) and IGF-1 (3.04-fold) levels. Stability testing demonstrated >90% retention of platelets and growth factors over 26 weeks. In vitro, FD-PRP accelerated wound closure (85% at 72 hours) compared to fresh PRP (70%) and saline (30%). In vivo, FD-PRP-treated wounds showed a mean size reduction of 56.17 % %by day 12, which was significantly higher than that in non-treated regions (23.5%). Conclusion: FD-PRP preserves platelet functionality and key growth factors, demonstrates excellent storage stability, and enhances wound healing both in vitro and in vivo. These findings support FD-PRP as a practical and effective alternative to fresh PRP for clinical applications that require standardized, off-the-shelf regenerative therapies. Keywords: Platelet-rich plasma, Freeze-drying, Lyophilization, Wound healing, Growth factors, Regenerative medicine.

Abstract: This study presents a comprehensive comparative assessment of the structural and functional responses of human serum and hen egg serum to natural aging (3, 5, and 7 days) and low thermal neutron fluxes (1.3×108, 2.16×108, and 3×108 N/cm2). Structural changes were investigated using Fourier transform infrared spectroscopy (FTIR), and biochemical parameters were used to assess functional changes. Natural aging induced progressive negative frequency shifts in the region of 1080-1340 cm-1 consistent with dehydration, increased hydrogen bonding, and decreased mobility of lipid and protein components. In contrast, neutron irradiation produced predominantly positive frequency shifts (+3 to +8 cm-1), with the most pronounced effects observed at an intermediate flux density of 2.16×108 N/cm2. These changes indicate a weakening of hydrogen bonds, a redistribution of electron density within amide and phosphate groups, and a partial reorganization of the protein-lipid matrix. Biochemical analysis correlates with spectroscopic observations: natural aging was associated with a decrease in total protein content and a moderate increase in ALT and AST activities, whereas neutron irradiation stimulated enzymatic activity and metabolic parameters in a nonlinear, flux-dependent manner. It is assumed that the observed non-monotonic reaction with a maximum effect at an intermediate neutron flux is caused by low-flux hormesis of thermal neutrons. Overall, the results show that low-flux neutron irradiation induces adaptive structural and functional responses in biological fluids that are fundamentally different from passive aging processes, highlighting the potential of IR spectroscopy as a sensitive tool for detecting molecular-level changes induced by low-flux neutron exposure. The observed non-monotonic responses are consistent with a hormetic-like interpretation, though further studies are needed to confirm this mechanism.

Abstract:

Indole-3-acetic acid (IAA) is the main natural auxin and a key regulator of plant growth. However, most commercial auxins are synthetically produced from non-renewable resources. Here, we present a minimal synthetic biology platform for microbial IAA production that also serves as a teaching model for genetic circuit design and bioprocess development. We developed codon-optimized versions of the iaaM and iaaH genes, which encode tryptophan 2-monooxygenase and indole-3-acetamide hydrolase, and assembled them into a compact expression cassette in Escherichia coli TOP10. Correct expression of both enzymes was confirmed by SDS-PAGE. The engineered strain was cultivated in a low-cost medium made from avocado seed hydrolysate, an agro-industrial waste, supplemented with tryptophan as a precursor. IAA levels of about 300 µg/mL were measured after 48 hours using the Salkowski assay and HPLC, with the medium costing five times less locally than traditional LB. The supernatants containing biosynthetic IAA induced strong root formation in tobacco leaf explants, confirming biological activity. Since this workflow follows the Design–Build–Test–Learn (DBTL) cycle: Design (pathway selection and codon optimization), Build (plasmid assembly), Test (protein expression, metabolite quantification, plant bioassays), and Learn (medium and process optimization), it provides a sustainable production method and an accessible educational platform for synthetic biology.

Abstract: Magnetizing living cells with superparamagnetic iron oxide nanoparticles (SPIONs) enables their remote manipulation using external magnetic field. This lays the foundation for magnetically assembling tissue precursors within cell-friendly, proliferation-permissive environments and holds considerable promise for biomedical applications, particularly in the development of complex single- and multicellular tissue constructs for bone and organ reconstruction. However, progress in this field is limited by the lack of robust mathematical tools for accurate control of ensembles of magnetic nano- and micro-objects. In practical printing scenarios, collective behavior and unavoidable statistical heterogeneity—such as variations in SPION size and shape or deviations in cell magnetization—render traditional equation-based modeling inadequate. We developed a hybrid modeling framework integrating conventional physics-based simulations with artificial intelligence–driven image analysis. Dynamic parameters were extracted from video recordings of magnetized cells moving within model microfluidic devices exposed to well-defined magnetic fields and gradients. The AI-based analysis enabled quantitative characterization of ensemble behavior under heterogeneous conditions. The proposed framework successfully captured the collective dynamics of magnetized cell ensembles and enabled accurate prediction and control of their spatial organization under external magnetic actuation. The integration of simulation and data-driven analysis provided robust parameter identification despite statistical heterogeneity within the system. This combined modeling approach offers a practical and effective tool for guiding the 3D magnetic assembly of living cells into functional tissue architectures. The framework advances the precision control of magnetically assisted bioprinting and supports future applications in bone and organ reconstruction.

Abstract: Background/objective: Approximately 90% of breast cancer–related deaths result from recurrence and metastasis. Emerging evidence indicates that tumor recurrence, invasion, and metastatic spread are strongly influenced by both the tumor microenvironment (TME) and the metastatic niche. M2 macrophages promote immune suppression, inhibit inflammation, and facilitate epithelial-to-mesenchymal transition, invasion, angiogenesis, and tumor progression—effects that are particularly pronounced in triple-negative breast cancer (TNBC). The objectives of this study were to develop engineered exosomes to selectively deplete M2 macrophages to delay the growth of primary tumor and distal metastasis and enhance overall survival. Methods: Engineered exosomes were developed using our invented platform to selectively target and deplete alternatively activated CD206⁺ M2 macrophages in primary and metastatic TMEs via antibody-dependent cell-mediated cytotoxicity (ADCC). Engineered exosomes were characterized for size, zeta potential, and successful incorporation of targeting peptides and proteins. Whole-body and tumor-specific biodistribution were assessed. In vitro and in vivo experiments were conducted to evaluate targeting specificity. Toxicity and immunogenicity were examined in immune-competent animal models. Two treatment paradigms were employed. Results: Engineered exosomes containing M2-macrophage targeting peptides and Fc-mIgG2b were successfully made and there were no significant size differences between engineered and control exosomes. Both in vitro and in vivo studies confirmed the specificity of the engineered exosomes. Biodistribution studies showed no significant uptake to the resident macrophages in the lung and liver. No significant immune activation, based on cytokine profiling, or organ-specific toxicity was observed in immune-competent models. Flowcytometry studies using splenocytes showed significant depletion of M2-macrophages following treatments with engineered exosomes, however, there was no effect on the distribution of T-cells. M2-targeting therapeutic exosomes significantly delayed the growth of primary tumors and metastatic lesions. Conclusion: These findings support the potential of precision exosome-based strategies for enhancing therapeutic outcomes in breast cancer.

Abstract: Milk microRNAs (miRNAs) are key mediators of maternal–offspring communication, potentially influencing postnatal development through systemic epigenetic regulation. While miRNAs in human and bovine milk are extensively studied, the regulatory landscape of camel milk remains underexplored despite its unique therapeutic reputation. In this study, we conducted a comparative cross-species analysis of miRNA targets from human (Homo sapiens), bovine (Bos taurus), and camel (Camelus dromedarius) milk. To estimate regulatory impact, we implemented the Global Score, a weighted metric integrating quantitative miRNA abundance with thermodynamic binding affinity (miRWalk 2.0), cross-validated by multi-algorithm statistical consensus via mirDIP 5.2.Analysis revealed distinct species-specific miRNA concentration strategies: human milk maintains a highly diversified "regulatory symphony" (90% mass distributed across 35 miRNAs), while camel milk utilizes a "biological intervention" strategy, with only 12 miRNAs constituting 90% of the pool and miR-148a exerting extreme dominance (61.8%). A conserved regulatory core (Common All) was identified, accounting for over 80% of the total regulatory weight, primarily associated with neuroplasticity and MAPK/Ras signaling.Camel milk miRNAs displayed a distinctive therapeutic signature, uniquely targeting pivotal immune and metabolic nodes, specifically STAT3 (linked to Th17 cell differentiation) and PPARD, providing a molecular basis for its reported anti-inflammatory and anti-diabetic effects. Conversely, human milk showed high-confidence human-specific targeting of NLGN3, essential for synaptic organization. Bovine milk exhibited a significantly closer functional alignment to the human "epigenetic template" (14.8% shared genes) than camel milk (1.5%), particularly in pathways maintaining genomic stability (e.g., XPO1). These findings demonstrate that milk miRNAs operate through both a universal developmental backbone and specialized regulatory environments, offering a comparative framework for applications in clinical nutrition and precision medicine.

Abstract:

Developmental engineering (DE) is a bottom-up strategy for generating functional tissues from modular tissues (MTs), overcoming limitations of conventional top-down approach. This study integrates theoretical simulations with empirical correlations to analyse microcarrier aggregation, oxygen transport, suspension conditions, and cell damage in stirred bioreactors, providing guidance for scalable MT production in DE. Microcarrier aggregates were modelled to evaluate minimum oxygen concentration (C_min). Results show that larger microcarrier diameters (d_mc) increase C_min because of longer diffusion distances. Aggregate geometry and packing configuration, including hexagonal close packing and the “kissing number,” influence oxygen limitation and explain observed C_min plateaus. Hydrodynamic behaviour was assessed using Zwietering correlation and Kolmogorov turbulence scaling. Denser microcarrier aggregates require higher minimum stirring speeds (N_min), while larger d_mc increases susceptibility to shear. Aggressive impeller designs and higher revolutions per minute reduce N_min but increase collision-induced cell damage. In contrast, higher medium density (e.g., 20% FBS) reduces shear stress and energy dissipation. A unified framework is proposed that integrates oxygen diffusion, aggregate geometry, microcarrier properties, and hydrodynamics to predict worst-case oxygen limitation and cell damage. The results clarify trade-offs between impeller design, agitation intensity, and aggregation, supporting scalable MT production using individual or aggregated modular scaffolds for DE-based tissue assembly.

Abstract: High-throughput omics technologies have transformed biology, enabling system-level exploration of genomes, transcriptomes, proteomes, metabolomes, and beyond. Despite progress, current omics approaches face limitations in temporal resolution, spatial context, and integrative interpretation. This review organizes established and emerging omics into a structured framework, highlighting conceptual innovations such as adaptomics, resiliomics, chrono-adaptomics, and signalomics. We discuss technological advances, computational strategies, and potential applications for research and clinical practice.

Abstract: The development of strategies and processes to valorize cheese whey is an area of constant advancement and growth, primarily due to the large volume generated daily and inherent technical difficulties in cost-effectively processing this material. In this regard, the present review, with a critical perspective from the authors, will discuss and analyze different strategies based on chromatographic innovations to develop processes that enable the direct and selective capture and purification of bovine lactoferrin (LF) and lactoperoxidase (LP) proteins from cheese whey. These proteins possess interesting commercial value and represent minor components of the whey protein fraction, with whey being their main source. The advancements achieved in terms of efficiency in the selectivity and purity obtained for the target proteins through the development of affinity chromatographic matrices and processes will be discussed, as well as the progress made in terms of productivity and whey processability.

Abstract:

Background: Leonotis ocymifolia and Laggera tomentosa are traditionally used in Ethiopian folk medicine to treat various ailments. This study evaluated the phytochemical constituents and antibacterial activity of their leaf extracts against selected human pathogenic bacteria. Methods: Crude extracts were prepared using six solvents (chloroform, ethanol, methanol, hexane, ethyl acetate, and water) via maceration. Phytochemical screening employed standard procedures. Plant activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae was assessed using disc diffusion (100 mg/mL), with MIC and MBC determinations. Results: L. ocymifolia contained flavonoids, tannins, alkaloids, and phenols; L. tomentosa additionally contained saponins. Chloroform extracts exhibited the highest activity, with inhibition zones of 20 ± 1 mm (L. ocymifolia against P. aeruginosa) and 20 ± 4.6 mm (L. tomentosa against P. aeruginosa). Water extracts showed minimal or no activity against several strains. MIC values ranged from 1.56–12.5 mg/mL (L. ocymifolia) and 3.13–6.25 mg/mL (L. tomentosa); MBC values ranged from 3.13–12.5 mg/mL and 3.13–6.25 mg/mL, respectively. Conclusion: Chloroform extracts of both plants demonstrated significant broad-spectrum antibacterial activity, validating their traditional use and highlighting their potential as sources of novel antibacterial agents.

Abstract: Astaxanthin can be derived from different sources such as petrochemical synthesis, natural sourcing from green algae or by microbial fermentation. As one of the strongest antioxidants known by nature, astaxanthin is rising attention as an active ingredient in cosmetical products to support the skin against oxidative stress. In contrast to widely performed chemical antioxidant activity assays, this study focuses on the comparison of synthetic, algal and corynebacterial astaxanthin in a physiological relevant test setting: the intracellular antioxidant activity in cultured human skin cells, keratinocytes. The astaxanthin-rich corynebacterial oleoresin demonstrated to be the superior antioxidant in that assay with a EC₅₀ of 2.7 µM whereas the synthetic and algal-based variants showed no significant effect. In terms of an application of such raw materials, it is therefore tempting to speculate that astaxanthin-containing corynebacterial oleoresins could serve as a natural and superior active ingredient for skin health applications in the future.

Abstract:

Polyhydroxyalkanoates (PHAs) are biodegradable microbial polyesters that represent a promising sustainable alternative to petroleum-based plastics. Salterns, hypersaline environments, are recognized as significant sources of halotolerant microorganisms that can produce PHAs in high-salinity conditions; however, Vietnamese saltern ecosystems have not been extensively investigated. This research aimed to isolate and initially characterize salt-tolerant bacteria capable of synthesizing PHAs from the Hon Khoi saltern in Khanh Hoa province, Vietnam. A total of 37 halotolerant bacterial isolates were obtained, and potential PHA-producing strains were initially screened using Sudan Black B and Nile Blue A. TEM microscopy was then employed to confirm the existence of PHA granules. Furthermore, FTIR spectroscopy and GC–MS/MS spectrometry were utilized to analyze the chemical structure and monomer composition of the extracted polymers. Six isolates were identified as PHA-producing bacteria, including Salinivibrio sp. HK101 and HK116, Halomonas sp. HK105, Priestia sp. HK125 and HK142, and Bacillus sp. HK130. These strains exhibited growth across 3–10% NaCl and temperatures from 25 to 45 °C. Priestia sp. HK142 and Salinivibrio sp. HK101 exhibited the most substantial PHA accumulation, achieving 50.72 ± 1.83% and 42.07 ± 1.8% of DCW, respectively. These results indicate that the Hon Khoi saltern represents a promising source of halotolerant PHA-producing bacteria with potential relevance for future biopolymer production studies.

Abstract: Thailand preserves one of the most extensive records of Mesozoic vertebrate footprints in Tropical Asia, yet these ichnological data have never been comprehensively synthesized. This review compiles and reassesses all known Triassic to Cretaceous vertebrate tracksites in Thailand to clarify their stratigraphic distribution, taxonomic diversity, and palaeobiogeographical significance. Published records, new field observations, and updated stratigraphic correlations are integrated to evaluate trackmaker attributions and temporal patterns. The Thai record documents diverse assemblages including chirotheriids, early theropods, sauropodomorphs, ornithopods, sauropods, and crocodilians. Upper Triassic–Lower Jurassic assemblages capture a major faunal transition, revealing the co-occurrence of non-dinosaurian archosaurs and some of the earliest dinosaurs in the region, whereas Lower Cretaceous sites are dominated by theropods, sauropods and diverse ornithopods. Comparison with other Asian ichnofaunas indicates faunal continuity across eastern Asia and supports early dinosaur dispersal into equatorial low latitudes. This synthesis also evaluates site conservation, highlighting the vulnerability of several Triassic localities and a positive trend of community-led discoveries since 2009, underscoring the need for proactive management and standardized digital documentation. Overall, the Thai ichnological succession represents the most complete Mesozoic footprint record presently known from Tropical Asia and provides key insights into vertebrate evolution, palaeoecology, and regional biogeography.

Abstract: Genome editing is widely used and conceptually simple, yet in practice, it is hindered by laborious workflows and high costs. These challenges stem from the difficulty of identifying and isolating cells that contain the desired user‑defined modifications, a problem compounded by the wide variability in editing efficiencies across cell types. While homology-directed repair (HDR) provides a mechanism for precise genome modification following nuclease-induced double-strand breaks (DSBs), it is frequently outcompeted by the dominant mutagenic non-homologous end-joining (NHEJ) pathway in mammalian cells. Therefore, we developed a novel enrichment method, Essential HDRescue, to increase the frequency of HDR events at a target site by co-targeting an essential genomic locus. Using both intrinsic positive and negative selection at a common essential gene, we enabled enrichment of precise editing events at a second, unlinked target site. We demonstrated that co-targeting essential genes in cancer cell lines and iPSCs increased HDR rates without the need for an exogenous reporter or selective drug. Analysis of resulting clones revealed that Essential HDRescue produced up to a 6‑fold increase in single‑allele edits and almost a four‑fold increase in homozygous edits relative to single‑targeted controls. By harnessing the intrinsic cellular dependencies that arise from DSB repair at essential loci, Essential HDRescue offers a widely applicable method to improve precise genome editing outcomes in mammalian cells, leaving only a minimal, protein-silent scar at the essential gene.

Abstract:

Background/Objectives: The escalating crisis of antibiotic resistance and the inherent limitations of conventional antibiotics necessitate the development of innovative therapeutic strategies. Targeted drug delivery (TDD) offers a powerful approach to enhance efficacy, minimize systemic toxicity, and circumvent bacterial resistance. This systematic review aims to evaluate the potential of unique bacterial transport systems (BTSs) and surface specific receptors as platforms for TDD via the "Trojan Horse" strategy (THS). Methods: A comprehensive literature review was conducted, focusing on studies that investigated the specificity and mechanisms of BTSs responsible for the uptake of essential metabolites. This includes an analysis of transport systems for siderophores, bacteria-specific sugars, cell wall components, D-amino acids, and vitamins. We assessed preclinical and clinical examples of drug conjugates utilizing these pathways, as well as emerging platforms such as bacteriophage-derived proteins, antibody-antibiotic conjugates, and bacterial extracellular vesicles (EVs). Results: BTSs demonstrate high specificity for their cognate substrates, providing effective molecular gateways for drug conjugate import. The siderophore-cephalosporin conjugate cefiderocol represents a clinically validated example, having received FDA approval. Preclinical studies further reveal that conjugates utilizing sugars (e.g., maltose, trehalose) and vitamins (e.g., B12) can significantly enhance antibiotic uptake and activity against both Gram-positive and Gram-negative pathogens, including drug-resistant strains. Emerging platforms like bacteriophage endolysins and engineered EVs show promise for overcoming biological barriers such as bacterial outer membranes and intracellular host niches. Conclusions: The THS leveraging BTSs represents a clinically viable and promising avenue for next-generation antibacterial therapies. While significant progress has been made, including regulatory approval of cefiderocol, further research is critically needed to identify novel BTSs, optimize drug-linker chemistry, improve the pharmacokinetics and biosafety of conjugates, and translate these innovative platforms into effective treatments for drug-resistant infections.