Optogenetics Illuminates Applications in Microbial Engineering.

Construction of microbial cell factories is a feasible strategy for the sustainable production of chemicals, biofuels, and pharmaceutical molecules. However, the complex metabolism and rigid regulation of microbes hinders the efficient synthesis of the products of interest. Proteomics and metabolomics analyze enzymes and metabolites in terms of systems biology, thus unraveling complex biological systems and providing significant clues for microbial metabolic engineering. Here, the applications of proteomics and metabolomics in microbial metabolic engineering were reviewed, including the construction of genome-scale metabolic models, optimization of microbial product synthesis, guidance of microbial stress tolerance engineering, and prediction of rate-limiting steps. In addition, proteomics and metabolomics could be employed to explore secondary metabolic pathways in plants, to reveal novel genes or pathways for microbial synthesis of natural products. Finally, the development of bio-big data was discussed.

Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells

Manipulating gene expression in vivo specifically in neurons with precise spatiotemporal control is important to study the function of gene(s) or pathway(s) in the nervous system. Although various transgenic approaches or virus-mediated transfection methods are available, they are time consuming and/or lack precise temporal control. Here we introduce an efficient electroporation approach to transfect adult dorsal root ganglion (DRG) neurons in vivo that enables manipulation of gene expression in an acute and precise fashion. We have applied this method to manipulate gene expression in three widely used in vivo models of axon injury and regeneration, including dorsal column transection, dorsal root rhizotomy, and peripheral axotomy. By electroporating DRGs with siRNAs against c-jun to specifically deplete c-Jun in adult neurons, we provide evidence for the role of c-Jun in regulation of in vivo axon regeneration. This method will serve as a powerful tool to genetically dissect axon regeneration in vivo.

The stimulator of interferon genes (STING) pathway is a central target in cancer immunotherapy, but current STING agonist therapies lack precision control, leading to suboptimal therapeutic outcomes and systematic adverse effects. Herein, we engineered a dual-locked immuno-polymeric nanoplatform (IPN) with precise spatiotemporal control over the release of STING agonists to enhance cancer immunotherapy. This platform, constructed from biocompatible poly(β-amino esters) (PBAE), incorporates the STING agonist (MSA-2) covalently linked via ester bonds, which is co-assembled with a sonosensitizer. Upon activation by ultrasound and natural esterase enzyme, IPN significantly enhances the localized release of MSA-2 within the tumor. Alongside, this platform augments the generation of toxic radicals, leading to the spread of tumor antigens and immunogenic biomolecules, subsequently initiating a high magnitude of antigen-specific T cells for tumor eradication. The multifaceted advantages of ultrasound and enzymes synergistically enhance the physical contact and spatial organization of immune-related reactants as well as chemical bioprocesses. This dual-locked IPN platform demonstrates an eight fold greater tumor inhibition compared to single-locked counterparts and a four fold enhancement over the summation effect, highlighting a safer and more effective paradigm for cancer immunotherapy.

Intracellular calcium release (ICR) is of fundamental importance for numerous physiological and pathological processes, from cell differentiation to neurotransmitter release and muscle contraction. The ability to precisely control ICR is crucial for understanding cellular signaling mechanisms and developing therapeutic interventions for calcium-related disorders. Light-induced ICR offers numerous advantages over conventional approaches, including superior specificity, precise spatiotemporal control, excellent reproducibility, and minimal invasiveness. Here, we demonstrate that cyanine dyes function as molecular jackhammers (MJHs) to induce ICR in human embryonic kidney (HEK) 293 and immortalized mouse myoblast C2C12 cells through vibronic-driven action (VDA) following red light (640 nm) activation. VDA generates longitudinally and axially coordinated whole-molecule vibrations, enabling mechanical interactions with cells. Structure-activity relationship studies of twenty synthesized MJHs demonstrated that sulfonate-containing derivatives most effectively triggered intracellular calcium release while exhibiting minimal cytotoxicity. Mechanistic studies showed that MJH-induced calcium release operates through the inositol-triphosphate (IP3) pathway rather than reactive oxygen species generation. Finally, we demonstrated light-activated MJH-induced calcium waves in vivo using transgenic Hydra vulgaris expressing a fluorescent calcium indicator. This work establishes MJHs as molecular-scale devices for remote control of cellular signaling, expanding the utility of cyanine dyes for modulating physiological processes with potential therapeutic applications.

Tumor progression, including metastasis, is significantly influenced by factors in the tumor microenvironment (TME) such as mechanical force, shear stress, chemotaxis, and hypoxia. At present, most cancer studies investigate tumor metastasis by conventional cell culture methods and animal models, which are limited in data interpretation. Although patient tissue analysis, such as human patient-derived xenografts (PDX), can provide important clinical relevant information, they may not be feasible for functional studies as they are costly and time-consuming. Thus, in vitro three-dimensional (3D) models are rapidly being developed that mimic TME and allow functional investigations of metastatic mechanisms and drug responses. One of those new 3D models is tumor-on-a-chip technology that provides a powerful in vitro platform for cancer research, with the ability to mimic the complex physiological architecture and precise spatiotemporal control. Tumor-on-a-chip technology can provide integrated features including 3D scaffolding, multicellular culture, and a vasculature system to simulate dynamic flow in vivo. Here, we review a select set of recent achievements in tumor-on-a-chip approaches and present potential directions for tumor-on-a-chip systems in the future for areas including mechanical and chemical mimetic systems. We also discuss challenges and perspectives in both biological factors and engineering methods for tumor-on-a-chip progress. These approaches will allow in the future for the tumor-on-a-chip systems to test therapeutic approaches for individuals through using their cancerous cells gathered through approaches like biopsies, which then will contribute toward personalized medicine treatments for improving their outcomes.

Ultrasound can activate nano/microparticles to induce reactive oxygen species (ROS). The advantages of deep penetration and precise spatiotemporal control are demonstrated for multiple applications, such as sonodynamic therapy, chemical industry, and environmental treatment. Meanwhile, a toolbox of inorganic particles is developed to enhance ROS production via cavitation enhancement, sonoluminescence, and piezocatalytic effect. Nonetheless, sophisticated influences of ultrasonic parameters hamper further exploration of novel sonosensitized materials. In this perspective, the influential parameters in different mechanisms are reviewed, emphasizing the relationship between ultrasound frequency and catalytic activity, and outlooks are provided on the study of inorganic sonosensitizers.

The field of drug delivery has significantly advanced tumor therapy, yet challenges such as the predictability of treatment outcomes and the real‐time monitoring of tumor progression, such as recurrence or metastasis. Luminescent metal–organic frameworks (L‐MOFs) are a promising solution to overcome these issues. This review offers a comprehensive exploration of the design and synthesis of L‐MOFs, presenting their crucial optical properties. The review highlights the pivotal role of L‐MOFs in enabling precision medicine for tumor treatment driven by their performance. Through strategically harnessing light‐manipulated metal–organic frameworks (MOFs), real‐time therapeutic monitoring and precise spatiotemporal control in tumor treatment can be achieved. Moreover, this review meticulously examines the influence of photomanipulating MOFs on in situ drug synthesis, introducing the catalytic role in light‐manipulated gas therapy. Additionally, the role of L‐MOFs as gene therapy vectors that utilize light to trigger gene expression responses is also comprehensively explored in the article, aiming to highlight their role in enabling tumor therapy through gene therapy. The review concludes by proposing an artificial intelligence (AI)‐mediated blueprint for tumor treatment with L‐MOFs, aiming to pave the way for innovative approaches through light manipulation.

Exploiting NF-κB transcription factor peptide conjugation, a Ru(II)-bis-tap complex (tap = 1,4,5,8-tetraazaphenanthrene) was targeted specifically to the nuclei of live HeLa and CHO cells for the first time. DNA binding of the complex within the nucleus of live cells was evident from gradual extinction of the metal complex luminescence after it had crossed the nuclear envelope, attributed to guanine quenching of the ruthenium emission via photoinduced electron transfer. Resonance Raman imaging confirmed that the complex remained in the nucleus after emission is extinguished. In the dark and under imaging conditions the cells remain viable, but efficient cellular destruction was induced with precise spatiotemporal control by applying higher irradiation intensities to selected cells. Solution studies indicate that the peptide conjugated complex associates strongly with calf thymus DNA ex-cellulo and gel electrophoresis confirmed that the peptide conjugate is capable of singlet oxygen independent photodamage to plasmid DNA. This indicates that the observed efficient cellular destruction likely operates via direct DNA oxidation by photoinduced electron transfer between guanine and the precision targeted Ru(II)-tap probe. The discrete targeting of polyazaaromatic complexes to the cell nucleus and confirmation that they are photocytotoxic after nuclear delivery is an important step toward their application in cellular phototherapy.

The cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway regulates diverse cellular processes through precise spatiotemporal control across subcellular compartments. Förster resonance energy transfer (FRET)-based A-kinase activity reporters (AKARs) have enabled live-cell visualization of PKA activity, but their limited dynamic range constrains detection of subtle or compartment-specific signaling events. Here, we present a suite of sixth-generation cyan/yellow FRET-based PKA sensors (the AKAR6 series) with substantially enhanced sensitivity and improved selectivity. Systematic optimization of the FRET donor-acceptor pair and sensor backbone yields superior performance versus previous best-in-class FRET-based AKARs, enabling robust detection of subtle PKA activity changes across diverse experimental modalities, including flow cytometry, fluorescence lifetime-based FRET, and two-photon imaging in brain slices. We further leverage kinome atlas data to engineer a variant with improved selectivity for more accurate visualization of nuclear PKA activity. Using the AKAR6 toolkit, we showed that in contrast to strong GPCR-induced PKA activities across all tested compartments in PC12 cells, growth factors stimulated a significant PKA activity at the trans-Golgi network but no detectable activity at cis-Golgi, signifying highly compartmentalized PKA signaling at the sub-organelle level. Furthermore, NGF and EGF induced sustained and transient PKA activity, respectively, across various intracellular compartments, including the nucleus, suggesting that growth factor-specific temporal controls are maintained across subcellular compartments. Together, the AKAR6 toolkit provides a sensitive, selective, and versatile platform for dissecting compartmentalized PKA signaling across cells and tissues.

Nucleic acid delivery offers tremendous potential for the treatment of acquired and hereditary diseases. Despite limited successes, the use of nucleic acid therapies has been hampered by the lack of safe and efficient delivery approaches. To address this challenge, Epps, Sullivan, and coworkers developed a new nucleic acid delivery framework predicated on a photo-responsive cationic block copolymer (BCP) that enabled tunable nucleic acid binding and precise spatiotemporal control over gene expression. This innovative platform, in which the polymer moieties directly responsible for nucleic acid complexation could be cleaved from the polymer upon photo-stimulation, significantly enhancing nucleic acid release. Furthermore, temporal control over polyplex disassembly facilitated the development of a simple, and potentially universal, kinetic modeling scheme for intracellular small interfering RNA (siRNA), messenger RNA, and protein concentrations, and that model was quantitatively validated using various genes across several animal cell lines and human primary cells. This versatile BCP-based framework easily accommodated: anionic excipients that increased siRNA potency by ~200% (on a per mass basis) over comparable polyplex systems; quantum dots that unlocked theranostic applications without impacting silencing performance; and small-molecule lipid co-formulations that enhanced transfection in human primary cells. Altogether, the system described herein shows great promise for the clinical translation of gene therapeutics. A new nucleic acid delivery framework, predicated on a photo-responsive cationic block copolymer (BCP), was used to precisely tune nucleic acid binding and provide spatiotemporal control over gene expression. This innovative platform leveraged a macromolecular design in which the polymer moieties directly responsible for nucleic acid complexation were cleaved from the polymer upon photo-stimulation, significantly enhancing nucleic acid release. Temporal control over polyplex disassembly facilitated development of a potentially universal, kinetic modeling scheme for gene silencing. Furthermore, this versatile BCP-based framework easily accommodated anionic excipients and quantum dot imaging constructs.

The clinical application of tumor vaccines is hindered by challenges such as time‐consuming and costly production processes. In this context, in situ cancer vaccines represent a promising strategy by leveraging endogenous tumor antigens to elicit robust antitumor T cell responses. Herein, a photoactivatable tumor‐targeting in situ nanovaccine, Lipo‐D8‐6, was constructed using cRGD‐functionalized liposomes that co‐encapsulated the photosensitizer chlorin e6 and a cleavable immunoadjuvant conjugate D8, allowing light‐triggered synchronous activation of three therapeutic modules. Upon near‐infrared light irradiation, Lipo‐D8‐6 generates reactive oxygen species that exert direct cytotoxicity on tumor cells and induce immunogenic cell death, while concurrently cleaving the responsive linker within D8 to achieve the controlled release of R848. In vivo biodistribution analysis confirmed the superior intratumoral accumulation of Lipo‐D8‐6, facilitating precise treatment. In a large‐volume tumor model, the nanovaccine exhibited pronounced antitumor efficacy, accompanied by enhanced tumor infiltration of CD8+ T cells. Overall, this work provides a simplified and effective approach for developing in situ nanovaccines that enable synergistic photodynamic immunotherapy with precise spatiotemporal control over immune activation.

As a non-invasive modality, sonodynamic therapy (SDT) offers several advantages in cancer treatment, including deep tissue penetration and precise spatiotemporal control, resulting from the interplay between low-intensity ultrasound and sonosensitizers. Piezoelectric materials, known for their remarkable capacity of interconversion of mechanical and electrical energy, have garnered considerable attention in biomedical applications, which can serve as pivotal sonosensitizers in SDT. These materials can generate internal electric fields via ultrasound-induced mechanical deformation, which modulates the alteration of charge carriers, thereby initiating surface redox reactions to generate reactive oxygen species (ROS) and realizing the therapeutic efficacy of SDT. This review provides an in-depth exploration of piezoelectric materials utilized in SDT, with a particular emphasis on recent innovations, elucidation of underlying mechanisms, and optimization strategies for advanced biomedical piezoelectric materials. Furthermore, the incorporation of piezoelectric sonosensitizers with immunotherapy, photodynamic, chemodynamic, and chemotherapy is explored, emphasizing their potential to enhance cancer therapy outcomes. By examining the basic principles of the piezoelectric effect and its contributions to SDT, this review sheds light on the promising applications of piezoelectric materials in oncology. It also highlights future directions for improving these materials and expanding their clinical utility in tumor sonodynamic therapy.

The role of internal and external stimuli in the rational design of skin-specific drug delivery systems

Synthesis and biological evaluation of a novel photo-activated histone deacetylase inhibitor