Super-resolution proximity labeling with enhanced direct identification of biotinylation sites.

Promiscuous labeling enzymes, such as APEX2 or TurboID, are commonly used in in situ biotinylation studies of subcellular proteomes or protein-protein interactions. Although the conventional approach of enriching biotinylated proteins is widely implemented, in-depth identification of specific biotinylation sites remains challenging, and current approaches are technically demanding with low yields. A novel method to systematically identify specific biotinylation sites for LC-MS analysis followed by proximity labeling showed excellent performance compared with that of related approaches in terms of identification depth with high enrichment power. The systematic identification of biotinylation sites enabled a simpler and more efficient experimental design to identify subcellular localized proteins within membranous organelles. Applying this method to the processing body (PB), a non-membranous organelle, successfully allowed unbiased identification of PB core proteins, including novel candidates. We anticipate that our newly developed method will replace the conventional method for identifying biotinylated proteins labeled by promiscuous labeling enzymes.

Charge Transfer-Promoted Excited State of a Heavy-Atom-Free Photosensitizer for Efficient Application of Mitochondria-Targeted Fluorescence Imaging and Hypoxia Photodynamic Therapy.

Conventional photosensitizers (PSs) used in photodynamic therapy (PDT) have shown preliminary success; however, they are often associated with several limitations including potential dark toxicity in healthy tissues, limited efficacy under acidic and hypoxic conditions, suboptimal fluorescence imaging capabilities, and nonspecific targeting during treatment. In response to these challenges, we developed a heavy-atom-free PS, denoted as Cz-SB, by incorporating ethyl carbazole into a thiophene-fused BODIPY core. A comprehensive investigation into the photophysical properties of Cz-SB was conducted through a synergistic approach involving experimental and computational investigations. The enhancement of intersystem crossing (kISC) and fluorescence emission (kfl) rate constants was achieved through a donor-acceptor pair-mediated charge transfer mechanism. Consequently, Cz-SB demonstrated remarkable efficiency in generating reactive oxygen species (ROS) under acidic and low-oxygen conditions, making it particularly effective for hypoxic cancer PDT. Furthermore, Cz-SB exhibited good biocompatibility, fluorescence imaging capabilities, and a high degree of localization within the mitochondria of living cells. We posit that Cz-SB holds substantial prospects as a versatile PS with innovative molecular design, representing a potential "one-for-all" solution in the realm of cancer phototheranostics.

Early Immune Remodeling Steers Clinical Response to First-Line Chemoimmunotherapy in Advanced Gastric Cancer.

Adding anti-programmed cell death protein 1 (anti-PD-1) to 5-fluorouracil (5-FU)/platinum improves survival in some advanced gastroesophageal adenocarcinomas (GEA). To understand the effects of chemotherapy and immunotherapy, we conducted a phase II first-line trial (n = 47) sequentially adding pembrolizumab to 5-FU/platinum in advanced GEA. Using serial biopsy of the primary tumor at baseline, after one cycle of 5-FU/platinum, and after the addition of pembrolizumab, we transcriptionally profiled 358,067 single cells to identify evolving multicellular tumor microenvironment (TME) networks. Chemotherapy induced early on-treatment multicellular hubs with tumor-reactive T-cell and M1-like macrophage interactions in slow progressors. Faster progression featured increased MUC5A and MSLN containing treatment resistance programs in tumor cells and M2-like macrophages with immunosuppressive stromal interactions. After pembrolizumab, we observed increased CD8 T-cell infiltration and development of an immunity hub involving tumor-reactive CXCL13 T-cell program and epithelial interferon-stimulated gene programs. Strategies to drive increases in antitumor immune hub formation could expand the portion of patients benefiting from anti-PD-1 approaches. SIGNIFICANCE: The benefit of 5-FU/platinum with anti-PD-1 in first-line advanced gastric cancer is limited to patient subgroups. Using a trial with sequential anti-PD-1, we show coordinated induction of multicellular TME hubs informs the ability of anti-PD-1 to potentiate T cell-driven responses. Differential TME hub development highlights features that underlie clinical outcomes. This article is featured in Selected Articles from This Issue, p. 695.

OrthoID: profiling dynamic proteomes through time and space using mutually orthogonal chemical tools.

Identifying proteins at organelle contact sites, such as mitochondria-associated endoplasmic reticulum membranes (MAM), is essential for understanding vital cellular processes, yet challenging due to their dynamic nature. Here we report "OrthoID", a proteomic method utilizing engineered enzymes, TurboID and APEX2, for the biotinylation (Bt) and adamantylation (Ad) of proteins close to the mitochondria and endoplasmic reticulum (ER), respectively, in conjunction with high-affinity binding pairs, streptavidin-biotin (SA-Bt) and cucurbit[7]uril-adamantane (CB[7]-Ad), for selective orthogonal enrichment of Bt- and Ad-labeled proteins. This approach effectively identifies protein candidates associated with the ER-mitochondria contact, including LRC59, whose roles at the contact site were-to the best of our knowledge-previously unknown, and tracks multiple protein sets undergoing structural and locational changes at MAM during mitophagy. These findings demonstrate that OrthoID could be a powerful proteomics tool for the identification and analysis of spatiotemporal proteins at organelle contact sites and revealing their dynamic behaviors in vital cellular processes.

Dual anticancer and antibacterial activity of fluorescent naphthoimidazolium salts.

Cancer has emerged as a significant global health challenge, ranking as the second leading cause of death worldwide. Moreover, cancer patients frequently experience compromised immune systems, rendering them susceptible to bacterial infections. Combining anticancer and antibacterial properties in a single drug could lead to improved overall treatment outcomes and patient well-being. In this context, the present study focused on a series of hydrophilic naphthoimidazolium salts with donor groups (NI-R), aiming to create dual-functional agents with antibacterial and anticancer activities. Among these compounds, NI-TPA demonstrated notable antibacterial activity, particularly against drug-resistant bacteria, with MIC value of 7.8 µg mL-1. Furthermore, NI-TPA exhibited the most potent cytotoxicity against four different cancer cell lines, with an IC50 range of 0.67-2.01 µg mL-1. The observed high cytotoxicity of NI-TPA agreed with molecular docking and dynamic simulation studies targeting c-Met kinase protein. Additionally, NI-TPA stood out as the most promising candidate for two-photo excitation, fluorescence bioimaging, and localization in lysosomes. The study findings open new avenues for the design and development of imidazolium salts that could be employed in phototheranostic applications for cancer treatment and bacterial infections.

Preparations of MgO Nanoparticles by a Poly(acrylic acid)s Template-Assisted Method and Photovoltaic Performances of Dye-Sensitized Solar Cells based on MgO lnterlayer.

Magnesium oxide (MgO) nanoparticles are commonly used to enhance the reactivity and performance of devices and systems in various applications, primarily due to the heat-resistance, binding, and alkaline properties of MgO. However, most of the methods used to synthesize MgO nanoparticles suffer from nonuniform particle size distributions that make it difficult to manufacture stable particles. In this study, uniform magnesium oxide (MgO) nanoparticles were developed for TiO2 photoelectrodes of dye-sensitized solar cells (DSSCs) to enhance their interfacial resistances. The uniform MgO nanoparticles were synthesized from MgO 93% using a poly(acrylic acid) template-assisted method. The particle size and crystalline structure of MgO nanoparticles were characterized by NANOPHOX particle size analysis, transmission electron microscopy, and X-ray diffraction. Multilayered TiO2 photoelectrodes containing interlayers of MgO nanoparticles were fabricated as photoelectrodes for DSSC devices, and their photovoltaic performances were investigated. When the MgO interlayer was introduced into the multilayered TiO2 photoelectrode, it not only increased the photocurrent value of the DSSC device but also improved its power conversion efficiency. The DSSC device containing the MgO interlayer and the scattering layer exhibited an open-circuit voltage of 0.74 V, a short-circuit current density of 14.60 mA/cm2, and a fill factor of 0.64 under a photointensity of 100 mW/cm2 at AM 1.5, resulting in an overall solar energy conversion efficiency of 6.94%. The application of an MgO interlayer in a DSSC device exhibited improved conductivity, charge transfer ability, and excellent device performance.

Imidazolium-Based Heavy-Atom-Free Photosensitizer for Nucleus-Targeted Fluorescence Bioimaging and Photodynamic Therapy.

The development of heavy-atom-free photosensitizers (PSs) for photodynamic therapy (PDT) has encountered significant challenges in achieving simultaneous high fluorescence emission and reactive oxygen species (ROS) generation. Moreover, the limited water solubility of these PSs imposes further limitations on their biomedical applications. To overcome these obstacles, this study presents a molecular design strategy employing hydrophilic heavy-atom-free PSs based on imidazolium salts. The photophysical properties of these PSs were comprehensively investigated through a combination of experimental and theoretical analyses. Notably, among the synthesized PSs, the ethylcarbazole-naphthoimidazolium (NI-Cz) conjugate exhibited efficient fluorescence emission (ΦF = 0.22) and generation of singlet oxygen (ΦΔ = 0.49), even in highly aqueous environments. The performance of NI-Cz was validated through its application in fluorescence bioimaging and PDT treatment in HeLa cells. Furthermore, NI-Cz holds promise for two-photon excitation and type I ROS generation, nucleus localization, and selective activity against Gram-positive bacteria, thereby expanding its scope for the design of heavy-atom-free PSs and phototheranostic applications.

How Do University Students Perceive Inequality, Relationships and Power in University Life in the COVID-19 Era?

This study examined university students' perceptions of inequality, relationships and power following the COVID-19 outbreak. We used a qualitative research method, inductive content analysis (ICA), to analyse their perceptions of inequality in their personal life, insiders and outsiders that show superiority in relationships and people with strong and weak characteristics of power structures. We extracted superordinate concepts, such as those in the individual, interaction and social/environmental dimensions, as the perceptions of inequality, insiders and outsiders and people with strong and weak characteristics. First, we found that university students experience inequalities when they perceive that individuals must cope independently with changes brought about by COVID-19. Second, the results showed that individuals can become insiders or outsiders depending on how they act during the COVID-19 pandemic. Finally, we demonstrated that strong individuals are less affected by COVID-19-related changes than weak individuals. Therefore, improving university students' quality of life requires plans based on the students' perceptions of inequality.

Photovoltaic Effects of Dye-Sensitized Solar Cells Using Double-Layered TiO2 Photoelectrodes and Pyrazine-Based Photosensitizers.

In this study, to obtain high performances of the dye-sensitized solar cells using the optimal TiO2 photoelectrode for the synthesized pyrazine-based organic photosensitizers, three types of TiO2 photoelectrodes were fabricated and evaluated for comparison. The double-layered nanoporous TiO2 photoelectrode (SPD type) consisted of a dispersed TiO2 layer and a transparent TiO2 layer. The single-layered nanoporous TiO2 photoelectrodes (D type and SP type) consisted of a dispersed TiO2 layer and a transparent TiO2 layer, respectively. The surface area, pore volume, and crystalline structures of the three types of TiO2 photoelectrodes were analyzed by Brunauer-Emmett-Teller method, field-emission scanning electron microscopy, and X-ray diffractometry to confirm their crystallinity and surface morphology. The structures of the three types of TiO2 photoelectrode-adsorbed organic sensitizers were investigated using X-ray photoelectron spectroscopy. The photovoltaic performances of DSSC devices using three organic photosensitizers adsorbed onto the three types of TiO2 photoelectrodes were investigated under a light intensity of 100 mW/cm2 at AM 1.5. The DSSC device using double-layered SPD type TiO2 photoelectrodes displayed 1.31∼2.64% efficiency, compared to single-layered SP type TiO2 photoelectrodes (1.31∼2.50%) and D type TiO2 photoelectrodes (0.90∼1.54%), using organic photosensitizers. The DSSC device using the SPD type TiO2 photoelectrode and trifluoromethylbenzopyrazine (TPPF) as a photosensitizer showed the highest performances: J sc of 5.69 mA/cm2, V oc of 0.69 V, FF of 0.67, and efficiency of 2.64%. The relationship between photovoltaic effects and interfacial resistance characteristics of DSSCs using the three organic photosensitizers adsorbed onto the three types of TiO2 photoelectrodes could be interpreted from interfacial resistances according to frequency through impedance analysis.

Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.

The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. We integrated optogenetic control into proximity labeling, a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. 'LOV-Turbo' works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffic between endoplasmic reticulum, nuclear and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from luciferase, enabling interaction-dependent proximity labeling. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.

Binding of USP4 to cortactin enhances cell migration in HCT116 human colon cancer cells.

Ubiquitin-specific protease 4 (USP4) is highly overexpressed in colon cancer and acts as a potent protooncogenic protein by deubiquitinating ß-catenin. However, its prominent roles in tumor formation and migration in cancer cells are not fully understood by its deubiquitinating enzyme (DUB) activity on ß-catenin. Thus, we investigated an additional role of USP4 in cancer. In this study, we identified cortactin (CTTN), an actin-binding protein involved in the regulation of cytoskeleton dynamics and a potential prognostic marker for cancers, as a new cellular interacting partner of USP4 from proximal labeling of HCT116 cells. Additionally, the role of USP4 in CTTN activation and promotion of cell dynamics and migration was investigated in HCT116 cells. We confirmed that interacting of USP4 with CTTN increased cell movement. This finding was supported by the fact that USP4 overexpression in HCT116 cells with reduced expression of CTTN was insufficient to promote cell migration. Additionally, we observed that USP4 overexpression led to a significant increase in CTTN phosphorylation, which is a requisite mechanism for cell migration, by regulating Src/focal adhesion kinase (FAK) binding to CTTN and its activation. Our results suggest that USP4 plays a dual role in cancer progression, including stabilization of ß-catenin as a DUB and interaction with CTTN to promote cell dynamics by inducing CTTN phosphorylation. Therefore, this study demonstrates that USP4 is important for cancer progression and is a good target for treating or preventing cancer.

Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.

The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions, and function with light. We integrated optogenetic control into proximity labeling (PL), a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the PL enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. "LOV-Turbo" works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffick between endoplasmic reticulum, nuclear, and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by BRET from luciferase, enabling interaction-dependent PL. Overall, LOV-Turbo increases the spatial and temporal precision of PL, expanding the scope of experimental questions that can be addressed with PL.

Imidazole-carbazole conjugate for two-photon-excited photodynamic therapy and fluorescence bioimaging.

A heavy-atom-free photosensitizer (CI) based on an imidazole-carbazole conjugate exhibited strong fluorescence emission and ROS generation via both type I and II mechanisms. In particular, CI showed efficient photodynamic therapy and fluorescence bioimaging under two-photon (TP) excitation (740 nm) toward HeLa cells with negligible dark toxicity.

Generation of novel oncolytic vaccinia virus with improved intravenous efficacy through protection against complement-mediated lysis and evasion of neutralization by vaccinia virus-specific antibodies.

BACKGROUND: Oncolytic virus immunotherapy has revolutionized cancer immunotherapy by efficiently inducing both oncolysis and systemic immune activation. Locoregional administration has been used for oncolytic virus therapy, but its applications to deep-seated cancers have been limited. Although systemic delivery of the oncolytic virus would maximize viral immunotherapy's potential, this remains a hurdle due to the rapid removal of the administered virus by the complement and innate immune system. Infected cells produce some vaccinia viruses as extracellular enveloped virions, which evade complement attack and achieve longer survival by expressing host complement regulatory proteins (CRPs) on the host-derived envelope. Here, we generated SJ-600 series oncolytic vaccinia viruses that can mimic complement-resistant extracellular enveloped virions by incorporating human CRP CD55 on the intracellular mature virion (IMV) membrane. METHODS: The N-terminus of the human CD55 protein was fused to the transmembrane domains of the six type I membrane proteins of the IMV; the resulting recombinant viruses were named SJ-600 series viruses. The SJ-600 series viruses also expressed human granulocyte-macrophage colony-stimulating factor (GM-CSF) to activate dendritic cells. The viral thymidine kinase (J2R) gene was replaced by genes encoding the CD55 fusion proteins and GM-CSF. RESULTS: SJ-600 series viruses expressing human CD55 on the IMV membrane showed resistance to serum virus neutralization. SJ-607 virus, which showed the highest CD55 expression and the highest resistance to serum complement-mediated lysis, exhibited superior anticancer activity in three human cancer xenograft models, compared with the control Pexa-Vec (JX-594) virus, after single-dose intravenous administration. The SJ-607 virus administration elicited neutralizing antibody formation in two immunocompetent mouse strains like the control JX-594 virus. Remarkably, we found that the SJ-607 virus evades neutralization by vaccinia virus-specific antibodies. CONCLUSION: Our new oncolytic vaccinia virus platform, which expresses human CD55 protein on its membrane, prolonged viral survival by protecting against complement-mediated lysis and by evading neutralization by vaccinia virus-specific antibodies; this may provide a continuous antitumor efficacy until a complete remission has been achieved. Such a platform may expand the target cancer profile to include deep-seated cancers and widespread metastatic cancers.

Structure and stability of polydiacetylene membrane systems: Molecular dynamics simulation studies.

We have performed full atomistic molecular dynamics (MD) simulations to investigate structure and stability of bilayer membrane systems consisting of monomeric or polymeric 10,12-pentacosadiynoic acid (PCDA) units connected with lysine groups by amide bonds. The PCDA monomer molecules show a twisted three-rod-domain structure with two kinks but upon polymerization, they possess more elongated conformation. The resulting polydiacetylene (PDA) membrane systems have stable membrane structures at room temperature, which is similar to biological lipid bilayer membranes and maintain their gel-like membrane integrity even up to as high as 370 K. Structural properties such as area per monomer, membrane thickness, density profile, 2D pair distribution function, and orientational correlation function are also calculated to understand the membrane structure and check its stability upon thermal fluctuation with atomistic resolution. This study is expected to provide the understanding about PDA membrane systems in atomistic details as well as significant insights into designing new novel PDA sensors.

A Study on Emotions to Improve the Quality of Life of South Korean Senior Patients Residing in Convalescent Hospitals.

This study examined the occurrence of emotion types and the contents and meanings of individual emotion types to improve the quality of life of South Korean senior patients in convalescent hospitals. This research is a sequential mixed study in which we conducted emotion frequency and content analyses with 20 elderly resident patients in a convalescent hospital. In the emotion frequency analysis, we performed emotion occurrence frequency analysis and clustering to create groups of subjects that showed similar distributions of emotions. The study results found that South Korean senior patients displayed six major emotions: joy, sorrow, anger, surprise, fear, and tranquility, including mixed emotional states. In the emotion content analysis, we used NVivo to categorize and analyze the interview contents based on emotion types. The study results show the characteristics of emotions according to patients' treatment and recovery, life within narrow boundaries, relationships with new people and family, and the appearances of themselves that they could not easily but must accept. In addition, these characteristics appeared in health, environment, relationships, and psychological structures. Ultimately, the study results suggest that improving the quality of life of South Korean senior patients requires understanding of their emotions and examining diverse emotions in multiple dimensions.

South Korean Early Cancer Patients' Perceptions of Difficulties in Fighting Their Disease: A Q Methodological Approach.

This study applied the Q methodology to explore breast cancer patients' perceived difficulties in their fight against the disease. We used literature analysis and in-depth interviews and selected 162 statements for the Q population. Then, we chose 40 universal and representative statements for the Q samples from the Q population. The P sample included 13 breast cancer patients in the early stage of the disease who participated in the Q sorting. We interviewed the study participants with high factor weights by type of P sample. The study's results showed three types of breast cancer patients' perceptions of difficulties in the initial fight against the disease. Type 1 showed 'fear of the future', Type 2 showed 'helplessness against what cannot be controlled', and Type 3 showed 'frustration due to difficulties in role performance.' Based on these results, we discuss the characteristics, meanings, and significance of individual types of breast cancer patients' perceptions of the disease, including suggestions for follow-up studies.

Perceptions of Smartphone App Use among Mothers Raising Young Children.

The purpose of this study was to categorize subjective perceptions of smartphone app use among mothers with young children, derive characteristics of each perception type, and interpret the findings to determine appropriate smartphone use for each perception type. We applied the Q methodology to classify and analyze the types of perceptions of mothers with young children. Ultimately, we selected 40 Q samples by comprehensively analyzing in-depth interviews with mothers of young children and conducting reviews of literature related to smartphone app use. The P samples in the study consisted of 31 mothers. We performed data analysis using the QUANL program. The analysis identified four types of smartphone users: (1) "multi-adapters" who actively utilize functions, (2) "chaos dilemma" users who understand the disadvantages of smartphones but cannot refrain from using smartphone apps, (3) "time-killer dependent" users who utilize apps to relieve temporary stress and anxiety, and (4) "self-development focused users" who pursue positive changes. Based on these findings, we suggest ways in which mothers of young children can utilize smartphone apps in a developmental and appropriate manner.

Photovoltaic Performances of Dye-Sensitized Solar Cells Based on Modified Polybutadiene Matrix Electrolytes by Sol-Gel Process.

A new type of polymer matrix electrolyte based on modified polybutadiene (modified PB) was developed for dye-sensitized solar cells (DSSCs) to improve their stability. The modified PB was fabricated by cross-linking the reaction of polybutadiene with siloxane groups as a substitute sol-gel process. A DSSC device using the modified PB matrix electrolyte showed an open-circuit voltage of 0.64 V, a short-circuit current density of 15.00 mA/cm2, and a fill factor of 0.58 under photointensity of 100 mW/cm2 at AM 1.5, consequently leading to an overall solar energy conversion efficiency of 5.49%. The DSSC device using the modified PB matrix electrolyte improved the conductivity, and the charge transfer ability showed the outstanding stability of the device.

Dual Molecular Design toward a Lysosome-Tagged AIEgen and Heavy-Atom-Free Photosensitizers for Hypoxic Cancer Photodynamic Therapy.

To date, a large number of photosensitizers (PS) have introduced heavy atoms to improve the ISC process and 1O2 generation. However, they often show low efficiency in hypoxic conditions, aggregate states, and turn-off PDT in the dark. Besides that, the toxicity of heavy metals is also concerned. Therefore, we developed lysosome-targeted heavy-metal-free PS (3S and 4S) based on thionated naphthalimide for hypoxic cancer photodynamic therapy (PDT), not only under white light but also in the dark via thermal-induced 1O2 generation. AIEgen (3O and 4O) were prepared for studying the PDT action of PSs (3S and 4S) in lysosome and aggregate state. We also examined the photophysical properties of AIEgen (3O and 4O) and PS (3S and 4S) by UV-vis absorption, fluorescent emission spectra, and theoretical calculations.