TRPV4 modulation affects mitochondrial parameters in adipocytes and its inhibition upregulates lipid accumulation.

Enhanced lipid-droplet formation by adipocytes is a complex process and relevant for obesity. Using knock-out animals, involvement of TRPV4, a thermosensitive ion channel in the obesity has been proposed. However, exact role/s of TRPV4 in adipogenesis and obesity remain unclear and contradictory. Here we used in vitro culture of 3T3L-1 preadipocytes and primary murine-mesenchymal stem cells as model systems, and a series of live-cell-imaging to analyse the direct involvement of TRPV4 exclusively at the adipocytes that are free from other complex signalling as expected in in-vivo condition. Functional TRPV4 is endogenously expressed in pre- and in mature-adipocytes. Pharmacological inhibition of TRPV4 enhances differentiation of preadipocytes to mature adipocytes, increases expression of adipogenic and lipogenic genes, enhances cholesterol, promotes bigger lipid-droplet formation and reduces the lipid droplet temperature. On the other hand, TRPV4 activation enhanced the browning of adipocytes with increased UCP-1 levels. TRPV4 regulates mitochondrial-temperature, Ca2+-load, ATP, superoxides, cardiolipin, membrane potential (ΔΨm), and lipid-mitochondrial contact sites. TRPV4 also regulates the extent of actin fibres, affecting the cells mechanosensing ability. These findings link TRPV4-mediated mitochondrial changes in the context of lipid-droplet formation involved in adipogenesis and confirm the direct involvement of TRPV4 in adipogenesis. These findings may have broad implication in treating adipogenesis and obesity in future.

Atom Efficient Synthesis of Trimethine Cyanines Using Formaldehyde as a Single Carbon Source.

In this work, we present an innovative and atom-efficient synthesis of trimethine cyanines (Cy3) using formaldehyde (FA) as a single-carbon reagent. The widespread application of Cy3 dyes in bioimaging and genomics/proteomics is often limited by synthetic routes plagued with low atom economy and substantial side-product formation. Through systematic investigation, we have developed a practical and efficient synthetic pathway for both symmetric and asymmetric Cy3 derivatives, significantly minimizing resource utilization. Notably, this approach yields water as the byproduct, in alignment with sustainable chemistry principles. Moreover, the efficient one-pot synthesis facilitates the detection of intracellular FA levels, utilizing the fluorescence signal of Cy3 in live cells. It is also possible to detect the endogenous FA in the intestinal tissues. We observed a significant decrease of FA in the small intestine of the inflammatory bowel disease (IBD) mice compared to the healthy mice. This methodological advancement not only enhances the scope of fluorescent dye synthesis but also contributes to the sustainable practices within chemical manufacturing, offering a significant leap forward in the development of environmentally friendly synthetic strategies.

Reduced dynamicity and increased high-order protein assemblies in dense fibrillar component of the nucleolus under cellular senescence.

Cellular senescence, which is triggered by various stressors, manifests as irreversible cell cycle arrest, resulting in the disruption of multiple nuclear condensates. One of the affected structures is the nucleolus, whose tripartite layout, separated into distinct liquid phases, allows for the stepwise progression of ribosome biogenesis. The dynamic properties of dense fibrillar components, a sub-nucleolar phase, are crucial for mediating pre-rRNA processing. However, the mechanistic link between the material properties of dense fibrillar components and cellular senescence remains unclear. We established a significant association between cellular senescence and alterations in nucleolar materiality and characteristics, including the number, size, and sphericity of individual subphases of the nucleolus. Senescent cells exhibit reduced fibrillarin dynamics, aberrant accumulation of high-order protein assemblies, such as oligomers and fibrils, and increased dense fibrillar component density. Intriguingly, the addition of RNA-interacting entities mirrored the diminished diffusion of fibrillarin in the nucleolus during cellular senescence. Thus, our findings contribute to a broader understanding of the intricate changes in the materiality of the nucleolus associated with cellular senescence and shed light on nucleolar dynamics in the context of aging and cellular stress.

TRPM8 affects relative "cooling and heating" of subcellular organelles in microglia in a context-dependent manner.

Thermoregulation and thermal homeostasis at the cellular and subcellular organelle level are poorly understood events. In this work, we used BV2, a microglial cell line, and a series of thermo-sensitive subcellular organelle-specific probes to analyze the relative changes in the spatio-temporal temperatures of different subcellular organelles, both qualitatively and quantitatively. These methodologies allowed us to understand the thermal relationship of different subcellular organelles also. We modulated BV2 cells by pharmacological application of activator or inhibitor of TRPM8 ion channel (a cold-sensitive ion channel) and/or by treating the cells with LPS, a molecule that induces pathogen-associated molecular patterns (PAMPs) signaling. We demonstrate that the temperatures of individual organelles remain variable within a physiological range, yet vary in different conditions. We also demonstrate that treating BV2 cells by TRPM8 modulators and/or LPS alters the organelle temperatures in a specific and context-dependent manner. We show that TRPM8 modulation and/or LPS can alter the relationship of mitochondrial membrane potential to mitochondrial temperature. Our work suggests that mitochondrial temperature positively influences ER temperature and negatively influences Golgi temperature. Golgi temperature positively influences membrane temperature. This understanding of thermal relationships may be crucial for dissecting cellular structures, function, and stress signaling and may be relevant for different diseases.

Development of a specific fluorescent probe to detect advanced glycation end products (AGEs).

Advanced glycation end products (AGEs) play a pivotal role in the aging process, regarded as a hallmark of aging. Despite their significance, the absence of adequate monitoring tools has hindered the exploration of the relationship between AGEs and aging. Here, we present a novel AGE-selective probe, AGO, for the first time. AGO exhibited superior sensitivity in detecting AGEs compared to the conventional method of measuring autofluorescence from AGEs. Furthermore, we validated AGO's ability to detect AGEs based on kinetics, demonstrating a preference for ribose-derived AGEs. Lastly, AGO effectively visualized glycation products in a collagen-based mimicking model of glycation. We anticipate that this study will enhance the molecular tool sets available for comprehending the physiological processes of AGEs during aging.

Development of a Mature B Lymphocyte Probe through Gating-Oriented Live-Cell Distinction (GOLD) and Selective Imaging of Topical Spleen.

B lymphocytes play a pivotal role in the adaptive immune system by facilitating antibody production. Young B cell progenitors originate in the bone marrow and migrate to the spleen for antigen-dependent maturation, leading to the development of diverse B cell subtypes. Thus, tracking B cell trajectories through cell type distinction is essential for an appropriate checkpoint assessment. Despite its significance, monitoring specific B cell subclasses in live states has been hindered by a lack of suitable molecular tools. In this study, we introduce CDoB as the first mature B cell-selective probe, enabling real-time discrimination of three classified stages in B-cell development: progenitor, transitional, and mature B cells, through a single analysis using CyTOF. The selective mechanism of CDoB, elucidated as gating-oriented live-cell distinction (GOLD), targets SLC25A16, identified through systematic screening of SLC-CRISPRa and CRISPRi libraries. CDoB selectively brightens mature B cells in the mitochondrial area using SLC25A16 as the main gate, and the staining intensity correlates positively with the expression level of SLC25A16 along the B cell maturation continuum. In spleen tissues, CDoB demonstrates selective marking in mature B cell areas in live tissue status, representing the first performance achieved by a small-molecule fluorescent probe.

Targeting Heme Oxygenase 2 (HO2) with TiNIR, a Theragnostic Approach for Managing Metastatic Non-Small Cell Lung Cancer.

Despite notable advancements in cancer therapeutics, metastasis remains a primary obstacle impeding a successful prognosis. Our prior study has identified heme oxygenase 2 (HO2) as a promising therapeutic biomarker for the aggressive subsets within tumor. This study aims to systematically evaluate HO2 as a therapeutic target of cancer, with a specific emphasis on its efficacy in addressing cancer metastasis. Through targeted inhibition of HO2 by TiNIR (tumor-initiating cell probe with near infrared), we observed a marked increase in reactive oxygen species. This, in turn, orchestrated the modulation of AKT and cJUN activation, culminating in a substantial attenuation of both proliferation and migration within a metastatic cancer cell model. Furthermore, in a mouse model, clear inhibition of cancer metastasis was unequivocally demonstrated with an HO2 inhibitor administration. These findings underscore the therapeutic promise of targeting HO2 as a strategic intervention to impede cancer metastasis, enhancing the effectiveness of cancer treatments.

Novel Fluorescent Strategy for Discriminating T and B Lymphocytes Using Transport System.

Fluorescent bioprobes are invaluable tools for visualizing live cells and deciphering complex biological processes by targeting intracellular biomarkers without disrupting cellular functions. In addition to protein-binding concepts, fluorescent probes utilize various mechanisms, including membrane, metabolism, and gating-oriented strategies. This study introduces a novel fluorescent mechanism distinct from existing ways. Here, we developed a B cell selective probe, CDrB, with unique transport mechanisms. Through SLC-CRISPRa screening, we identified two transporters, SLCO1B3 and SLC25A41, by sorting out populations exhibiting higher and lower fluorescence intensities, respectively, demonstrating contrasting activities. We confirmed that SLCO1B3, with comparable expression levels in T and B cells, facilitates the transport of CDrB into cells, while SLC25A41, overexpressed in T lymphocytes, actively exports CDrB. This observation suggests that SLC25A41 plays a crucial role in discriminating between T and B lymphocytes. Furthermore, it reveals the potential for the reversible localization of SLC25A41 to demonstrate its distinct activity. This study is the first report to unveil a novel strategy of SLC by exporting the probe. We anticipate that this research will open up new avenues for developing fluorescent probes.

Discovery of Live Cell Selective Fluorescent Probes and Elucidation of Their Mechanisms: Case Study of B Cell Selective Probe CDgB.

The development of small-molecule fluorescent probes for specific immune cell identification offers an economical alternative to expensive antibodies. Moreover, it enables the identification of live target cells and provides insights into the distinct properties of cells, leveraging their specific staining mechanisms. This chapter presents a comprehensive elucidation of the methodology employed for screening fluorescent compounds using flow cytometry measurements. A novel analytical approach is proposed to distinguish a fluorescent compound with a specific carbon length for B lymphocytes, involving an assessment of the staining index and the predominant ratio of immune cells. Moreover, a protocol is presented for investigating the staining mechanisms of these probes by employing cell mimicking models such as small unilamellar vesicles (SUVs).

The Effect of 3-Dimensional-Printed Sequential Dual Drug-Releasing Patch on the Capsule Formation Around the Silicone Implant in a Rat Model.

BACKGROUND: Implant-based breast reconstruction is associated with increased risk of early infection and late-stage capsular contracture. OBJECTIVES: We evaluated the feasibility of a dual drug-releasing patch that enabled the controlled delivery of antibiotics and immunosuppressants in a temporally and spatially appropriate manner to the implant site. METHODS: The efficacy of a dual drug-releasing patch, which was 3-dimensional-printed (3D-printed) with tissue-derived biomaterial ink, was evaluated in rats with silicone implants. The groups included implant only (n = 10); implant plus bacterial inoculation (n = 14); implant, bacterial inoculation, and patch loaded with gentamycin placed on the ventral side of the implant (n = 10), and implant, bacterial inoculation, and patch loaded with gentamycin and triamcinolone acetonide (n = 9). Histologic and immunohistochemical analyses were performed 8 weeks after implantation. RESULTS: The 2 drugs were sequentially released from the dual drug-releasing patch and exhibited different release profiles. Compared to the animals with bacterial inoculation, those with the antibiotic-only and the dual drug-releasing patch exhibited thinner capsules and lower myofibroblast activity and inflammation, indicating better tissue integration and less foreign body response. These effects were more pronounced with the dual drug-releasing patch than with the antibiotic-only patch. CONCLUSIONS: The 3D-printed dual drug-releasing patch effectively reduced inflammation and capsule formation in a rat model of silicone breast reconstruction. The beneficial effect of the dual drug-releasing patch was better than that of the antibiotic-only patch, indicating its therapeutic potential as a novel approach to preventing capsular contracture while reducing concerns of systemic side effects.

NeuM: A Neuron-Selective Probe Incorporates into Live Neuronal Membranes via Enhanced Clathrin-Mediated Endocytosis in Primary Neurons.

The development of a small-molecule probe designed to selectively target neurons would enhance the exploration of intricate neuronal structures and functions. Among such probes, NeuO stands out as the pioneer and has gained significant traction in the field of research. Nevertheless, neither the mechanism behind neuron-selectivity nor the cellular localization has been determined. Here, we introduce NeuM, a derivative of NeuO, designed to target neuronal cell membranes. Furthermore, we elucidate the mechanism behind the selective neuronal membrane trafficking that distinguishes neurons. In an aqueous buffer, NeuM autonomously assembles into micellar structures, leading to the quenching of its fluorescence (Φ=0.001). Upon exposure to neurons, NeuM micelles were selectively internalized into neuronal endosomes via clathrin-mediated endocytosis. Through the endocytic recycling pathway, NeuM micelles integrate into neuronal membrane, dispersing fluorescent NeuM molecules in the membrane (Φ=0.61). Molecular dynamics simulations demonstrated that NeuM, in comparison to NeuO, possesses optimal lipophilicity and molecular length, facilitating its stable incorporation into phospholipid layers. The stable integration of NeuM within neuronal membrane allows the prolonged monitoring of neurons, as well as the visualization of intricate neuronal structures.

Distinct chemical environments in biomolecular condensates.

Diverse mechanisms have been described for selective enrichment of biomolecules in membrane-bound organelles, but less is known about mechanisms by which molecules are selectively incorporated into biomolecular assemblies such as condensates that lack surrounding membranes. The chemical environments within condensates may differ from those outside these bodies, and if these differed among various types of condensate, then the different solvation environments would provide a mechanism for selective distribution among these intracellular bodies. Here we use small molecule probes to show that different condensates have distinct chemical solvating properties and that selective partitioning of probes in condensates can be predicted with deep learning approaches. Our results demonstrate that different condensates harbor distinct chemical environments that influence the distribution of molecules, show that clues to condensate chemical grammar can be ascertained by machine learning and suggest approaches to facilitate development of small molecule therapeutics with optimal subcellular distribution and therapeutic benefit.

Chronological development of functional fluorophores for bio-imaging.

Functional fluorophores represent an emerging research field, distinguished by their diverse applications, especially in sensing and cellular imaging. After the discovery of quinine sulfate and subsequent elucidation of the fluorescence mechanism by Sir George Stokes, research in the field of fluorescence gained momentum. Over the past few decades, advancements in sophisticated instruments, including super-resolution microscopy, have further promoted cellular imaging using traditional fluorophores. These advancements include deciphering sensing mechanisms via photochemical reactions and scrutinizing the applications of fluorescent probes that specifically target organelles. This approach elucidates molecular interactions with biomolecules. Despite the abundance of literature illustrating different classes of probe development, a concise summary of newly developed fluorophores remains inadequate. In this review, we systematically summarize the chronological discovery of traditional fluorophores along with new fluorophores. We briefly discuss traditional fluorophores ranging from visible to near-infrared (NIR) in the context of cellular imaging and in vivo imaging. Furthermore, we explore ten new core fluorophores developed between 2007 and 2022, which exhibit advanced optical properties, providing new insights into bioimaging. We illustrate the utilization of new fluorophores in cellular imaging of biomolecules, such as reactive oxygen species (ROS), reactive nitrogen species (RNS), and proteins and microenvironments, especially pH and viscosity. Few of the fluorescent probes provided new insights into disease progression. Furthermore, we speculate on the potential prospects and significant challenges of existing fluorophores and their potential biomedical research applications. By addressing these aspects, we intend to illuminate the compelling advancements in fluorescent probe development and their potential influence across various fields.

Mitochondrial temperature homeostasis resists external metabolic stresses.

Based on studies with a fluorescent reporter dye, Mito Thermo Yellow (MTY), and the genetically encoded gTEMP ratiometric fluorescent temperature indicator targeted to mitochondria, the temperature of active mitochondria in four mammalian and one insect cell line was estimated to be up to 15°C above that of the external environment to which the cells were exposed. High mitochondrial temperature was maintained in the face of a variety of metabolic stresses, including substrate starvation or modification, decreased ATP demand due to inhibition of cytosolic protein synthesis, inhibition of the mitochondrial adenine nucleotide transporter and, if an auxiliary pathway for electron transfer was available via the alternative oxidase, even respiratory poisons acting downstream of oxidative phosphorylation (OXPHOS) complex I. We propose that the high temperature of active mitochondria is an inescapable consequence of the biochemistry of OXPHOS and is homeostatically maintained as a primary feature of mitochondrial metabolism.

Development of Iodinated Indocyanine Green Analogs as a Strategy for Targeted Therapy of Liver Cancer.

Liver cancer is one of the leading causes of cancer-related deaths, with a significant increase in incidence worldwide. Novel therapies are needed to address this unmet clinical need. Indocyanine green (ICG) is a broadly used fluorescence-guided surgery (FGS) agent for liver tumor resection and has significant potential for conversion to a targeted therapy. Here, we report the design, synthesis, and investigation of a series of iodinated ICG analogs (I-ICG), which can be used to develop ICG-based targeted radiopharmaceutical therapy. We applied a CRISPR-based screen to identify the solute carrier transporter, OATP1B3, as a likely mechanism for ICG uptake. Our lead I-ICG compound specifically localizes to tumors in mice bearing liver cancer xenografts. This study introduces the chemistry needed to incorporate iodine onto the ICG scaffold and defines the impact of these modifications on key properties, including targeting liver cancer in vitro and in vivo.

The Lysosomal Calcium Channel TRPML1 Maintains Mitochondrial Fitness in NK Cells through Interorganelle Cross-Talk.

Cytotoxic lymphocytes eliminate cancer cells through the release of lytic granules, a specialized form of secretory lysosomes. This compartment is part of the pleomorphic endolysosomal system and is distinguished by its highly dynamic Ca2+ signaling machinery. Several transient receptor potential (TRP) calcium channels play essential roles in endolysosomal Ca2+ signaling and ensure the proper function of these organelles. In this study, we examined the role of TRPML1 (TRP cation channel, mucolipin subfamily, member 1) in regulating the homeostasis of secretory lysosomes and their cross-talk with mitochondria in human NK cells. We found that genetic deletion of TRPML1, which localizes to lysosomes in NK cells, led to mitochondrial fragmentation with evidence of collapsed mitochondrial cristae. Consequently, TRPML1-/- NK92 (NK92ML1-/-) displayed loss of mitochondrial membrane potential, increased reactive oxygen species stress, reduced ATP production, and compromised respiratory capacity. Using sensitive organelle-specific probes, we observed that mitochondria in NK92ML1-/- cells exhibited evidence of Ca2+ overload. Moreover, pharmacological activation of the TRPML1 channel in primary NK cells resulted in upregulation of LC3-II, whereas genetic deletion impeded autophagic flux and increased accumulation of dysfunctional mitochondria. Thus, TRPML1 impacts autophagy and clearance of damaged mitochondria. Taken together, these results suggest that an intimate interorganelle communication in NK cells is orchestrated by the lysosomal Ca2+ channel TRPML1.

Menthol causes mitochondrial Ca2+-influx, affects structure-function relationship and cools mitochondria.

Menthol is a small bioactive compound able to cause several physiological changes and has multiple molecular targets. Therefore, cellular response against menthol is complex, and still poorly understood. In this work, we used a human osteosarcoma cell line (Saos-2) and analysed the effect of menthol, especially in terms of cellular, subcellular and molecular aspects. We demonstrate that menthol causes increased mitochondrial Ca2+ in a complex manner, which is mainly contributed by intracellular sources, including ER. Menthol also changes the Ca2+-load of individual mitochondrial particles in different conditions. Menthol increases ER-mito contact points, causes mitochondrial morphological changes, and increases mitochondrial ATP, cardiolipin, mitochondrial ROS and reduces mitochondrial membrane potential (ΔΨm). Menthol also prevents the mitochondrial quality damaged by sub-lethal and lethal doses of CCCP. In addition, menthol lowers the mitochondrial temperature within cell and also serves as a cooling agent for the isolated mitochondria in a cell free system too. Notably, menthol-induced reduction of mitochondrial temperature is observed in diverse types of cells, including neuronal, immune and cancer cells. As the higher mitochondrial temperature is a hallmark of several inflammatory, metabolic, disease and age-related disorders, we propose that menthol can serve as an active anti-aging compound against all these disorders. These findings may have relevance in case of several pharmacological and clinical applications of menthol. SIGNIFICANCE STATEMENT: Menthol is a plant-derived bioactive compound that is widely used for several physiological, behavioural, addictive, and medicinal purposes. It is a well-established "cooling and analgesic agent". However, the exact cellular and sub-cellular responses of menthol is poorly understood. In this work, we have characterized the effects of menthol on mitochondrial metabolism. Menthol regulates mitochondrial Ca2+, ATP, superoxides, cardiolipin, membrane-potential, and ER-mito contact sites. Moreover, the cooling agent menthol also cools down mitochondria and protects mitochondrial damage by certain toxins. These findings may promote use of menthol as a useful supplementary agent for anti-aging, anti-cancer, anti-inflammatory purposes where higher mitochondrial temperature is prevalent.

Improved tropoelastin synthesis in the skin by codon optimization and nucleotide modification of tropoelastin-encoding synthetic mRNA.

Loss of elastin due to aging, disease, or injury can lead to impaired tissue function. In this study, de novo tropoelastin (TE) synthesis is investigated in vitro and in vivo using different TE-encoding synthetic mRNA variants after codon optimization and nucleotide modification. Codon optimization shows a strong effect on protein synthesis without affecting cell viability in vitro, whereas nucleotide modifications strongly modulate translation and reduce cell toxicity. Selected TE mRNA variants (3, 10, and 30 µg) are then analyzed in vivo in porcine skin after intradermal application. Administration of 30 µg of native TE mRNA with a me1 Ψ modification or 10 and 30 µg of unmodified codon-optimized TE mRNA is required to increase TE protein expression in vivo. In contrast, just 3 µg of a codon-optimized TE mRNA variant with the me1 Ψ modification is able to increase protein expression. Furthermore, skin toxicity is investigated in vitro by injecting 30 µg of mRNA of selected TE mRNA variants into a human full-thickness skin model, and no toxic effects are observed. Thereby, for the first time, an increased dermal TE synthesis by exogenous administration of synthetic mRNA is demonstrated in vivo. Codon optimization of a synthetic mRNA can significantly increase protein expression and therapeutic outcome.

NMN sensor cocktail: selective sensing of nicotinamide mononucleotide over citric acid.

The fluorescent probe pair, NBD-B2 and Styryl-51F, selectively detects NMN over citric acid. NBD-B2 exhibits increased fluorescence, while Styryl-51F shows decreased fluorescence upon NMN addition. Their ratiometric fluorescence change enables highly sensitive and wide-range detection of NMN, effectively distinguishing it not only from citric acid but also other NAD boosters.