Toxoplasma gondii is an intracellular eukaryotic parasite in the phylum Apicomplexa. Reversible, conditional control of gene transcription was achieved previously in this organism using a tet-off system that was controlled by tetracycline. Although revolutionary for the field at the time, that system in Toxoplasma yielded modest signal-to-noise ratios (SNR) and proved to be leaky in the "off" state (i.e., it suffered from high background expression). Here we report the development of a new reversible and robust Conditional Transcription Factor (CTF) that is controlled by rapamycin. Although we originally intended to design a dimerizable drug-on transcription control system, the CTF unexpectedly functions in a drug-off fashion. In stably-transfected tachyzoites, CTF regulates a fluorescent reporter gene in Toxoplasma to achieve almost no background expression in the presence of rapamycin (i.e. near-zero leakiness), while in the absence of rapamycin 84% of tachyzoites express EYFP in comparison with 87% of constitutively-expressed positive controls, with 59% mean fluorescence intensity relative to positive controls. Conditional regulation of the median fluorescence intensity of the reporter gene reached an impressive SNR averaging 1489, approximating an "on/off" switch. The modular design of the CTF is expected to facilitate application to varied genes, incremental modifications, and adaptation for use in other apicomplexan organisms.

Fluorescent reporter systems for protein expression enable real-time, dynamic, and single-cell visualization of gene expression in living cells, but lack sufficient sensitivity for detecting low-abundance proteins. Herein, we developed a signal-amplified split superfolder green fluorescent protein (sfGFP) reporter system: sfGFP was split into the GFP1-10 fragment driven by a constitutively strong promoter and a 20× tandem GFP11 tag controlled by weak promoters, with the incorporation of coiled-coil dimerization domains to facilitate fragment complementation. This design enhanced fluorescent signals by approximately 10-fold, significantly improving detection sensitivity and enabling clear visualization of green fluorescence even at extremely low mRNA levels. Application of this system to evaluate Tet-On inducible systems revealed substantial basal leakage in Tet-On 2G. Moreover, real-time monitoring of endogenous weakly promoters (e.g., VIM, HPRT1, GUSB) demonstrated that the fluorescent intensity was approximately 10-fold higher than that of conventional reporter systems. This platform provides a highly sensitive and versatile tool for weak promoter activity analysis, dynamic tracking of low-abundance genes, and investigation of cell types and states.

Abscisic acid (ABA) is a sesquiterpenoid plant hormone widely used in agriculture and the food industry. In Yarrowia lipolytica, ABA production relies on complex heterologous enzymatic pathways that have not been systematically engineered, which severely limits biosynthetic efficiency. Here, we establish de novo ABA biosynthesis in Y. lipolytica. We optimized carbon flux by up- and down-regulating key genes, and rationally engineered rate-limiting cytochrome P450 monooxygenases BcABA2 to improve pathway efficiency. Electron transfer was engineered by incorporating an NADPH regeneration module to enhance BcABA2 catalysis. Peroxisomal compartmentalization of the entire ABA pathway, followed by complementation of the defective tag to obtain a complete strain, increased ABA titer to 330.69mg/L. The engineered strain was scaled up in a 5 L bioreactor, achieving an ABA titer of 2554.36mg/L after 168 h, which is the highest titer reported to date for microbial fermentation. This work provides feasible enzyme and metabolic engineering strategies for microbial cell factory production of ABA and other terpenoids.

Zeolitic imidazolate framework-8 (ZIF-8) has limitations in vaccine delivery, including poor targeting and rapid clearance. The outer membrane phosphoporin pore protein (PhoE) has been validated as a promising antigen against Klebsiella pneumoniae. We developed a mannose (M)-functionalized ZIF-8 nanoparticle (PhoE@M-ZIF-8) via polyethylene glycol (PEG) conjugation and mannose conjugation to enable targeted delivery to antigen-presenting cells (APCs). PhoE@M-ZIF-8 exhibited pH-responsive antigen release and was internalized via mannose receptor-mediated macropinocytosis, promoting dendritic cell maturation (CD11c+CD86+ upregulation) and inducing a balanced T helper type 1/type 2 (Th1/Th2) immune response. In vivo, PhoE@M-ZIF-8 achieved potent protection against lethal Klebsiella pneumoniae challenge with only two immunizations, showing high survival rates and favorable biosafety. This work presents a versatile APC-targeting platform for effective subunit vaccine development.

Acute pancreatitis (AP)-induced intestinal barrier disruption drives fatal systemic complications. We engineered human umbilical cord mesenchymal stem cell-derived extracellular vesicles (hUC-MSC-EVs) to deliver emodin-a bioactive compound limited by poor bioavailability-for targeted intestinal protection. In TNF-α-stimulated intestinal epithelial cells (CCD-841CON), EV-loaded emodin (EVs-Emodin) synergistically suppressed NLRP3 inflammasome activation, pyroptosis, reactive oxygen species production, and inflammatory cytokines, outperforming monotherapies. EVs-Emodin restored cell viability and curtailed apoptosis. In taurocholate-induced AP mice, intravenous EVs-Emodin attenuated systemic inflammation, promoted the expression of Occludin and ZO-1, mitigated intestinal tissue lesions, promoted epithelial regeneration, inhibited inflammasome activation, and alleviated mitochondrial damage. hUC-MSC-EVs overcome emodin's delivery limitations, providing a synergistic strategy to protect the intestinal barrier via NLRP3/pyroptosis inhibition and oxidative stress mitigation, offering a promising therapeutic approach for AP-associated intestinal injury.

Molecular genetics has propelled advancements in scientific instrumentation, yielding transformative discoveries from CRISPR-mediated gene editing and detailed protein identification to sophisticated biosensor fabrications. This profound shift has also reshaped the landscape of live attenuated vaccine (LAV) development in contrast to conventional methods. Here we explore how modern molecular strategies have superseded previous empirical approaches, moving toward deliberate genetic modifications that both enhance and balance the aspects of LAV safety, stability, and potent immunogenicity. By detailing the transition from classical approaches to targeted, molecular-driven attenuation, our work reviews how advanced genetic methods address historical limitations and expand the potential for vaccine design. Hence, the current paper bridges the gap between foundational vaccine practices and cutting-edge biotechnology, offering a comprehensive perspective on the progression of molecular genetic strategies on LAV development and its future trajectory. The paper also elaborates on the key challenges of raising LAVs to clinical standards while describing in tandem the genetic approaches to overcome the limitations. Critical performance factors governing the LAV market and clinical deployment, alongside the pivotal role of artificial intelligence in refining LAV rational design, are also further discussed.

Phospholipid-based nanocarriers are an adaptable and chemically tunable class of drug delivery systems that self-assemble into bilayered vesicles due to the amphiphilic nature of phospholipids. Phospholipid-based nanocarriers can encapsulate both hydrophilic and hydrophobic drugs through non-covalent interactions and modulation of lipid phase behavior. This review explores the molecular and supramolecular principles governing the formation, stability, and function of key phospholipid-derived nanocarriers, including liposomes, transferosomes, ethosomes, invasomes, phytosomes, pharmacosomes, and virosomes. The structural attributes-such as bilayer packing, surface charge, curvature elasticity, and membrane permeability-are critically evaluated for the impact of those structural parameters on optimizing drug loading, drug release, and bioavailability. For example, variations in bilayer packing affect the encapsulation efficiency and drug release profiles, while surface charge modulates cellular uptake and colloidal stability. Curvature elasticity plays a pivotal role in membrane fusion and drug release, and membrane permeability determines the rate at which drugs diffuse from the nanocarrier. Emerging multilamellar systems such as vesosomes and spongosomes are also discussed for their potential in site-specific, controlled drug release. Common fabrication techniques (e.g., thin-film hydration, ethanol injection, freeze-thaw, and microfluidics) and characterization methods (e.g., DLS, DSC, FTIR, and cryo-TEM) are reviewed. The translational landscape is assessed through clinically approved liposomal drugs, patent trends, and ongoing trials involving stimuli-responsive systems. Challenges related to colloidal stability, tumor penetration, immune interactions, and large-scale manufacturing are addressed. This review provides a chemistry-centered framework to guide the rational design and clinical development of phospholipid nanocarriers, particularly in cancer therapeutics.

Hyaluronic acid (HA) is an important homopolysaccharide also known as a natural anionic mucopolysaccharide or glycosaminoglycan formed by the alternate connection of glucuronic acid and N-acetylglucosamine as disaccharide units. For this review, the literature was searched in PubMed, Scopus, and Web of Science, using a few specific keywords including hyaluronic acid, biopolymer, glucuronic acid, N-acetylglucosamine, cosmetic, pharmaceutical, anti-aging and wound. Among the searched publications, further screening was done to select reports on the use of HA in healthcare and pharmaceutical products. Information relevant to the topic of this review has been presented in the article using 100 selected references, in eight tables and five figures. HA is a commercially valuable biocompatible polymer, which has been traditionally derived from rooster combs and animal tissues. However, it is currently produced through microbial fermentation by group C Streptococci. This biopolymer occurs naturally in several human tissues, including the skin and eyes. HA is a vital ingredient used in numerous products by global industries due to its unique characteristics, like inherent biocompatibility, tissue regenerative capacity, and anti-inflammatory properties. There has been a significant interest in recent years, enhanced across the pharmaceutical and cosmetic industries. Equally, the increased consumer interest from the aging population is compelling the demand for anti-aging products. Therefore, the global market for products formulated with HA marketed with defined benefits is experiencing significant growth. In this review, we have summarized current commercial applications of HA in many industries and consequently the increasing market size in specific application sectors in the next five years by 2030 in the international market.