Search Results (360)

The amygdala is composed of several nuclei, including the lateral nucleus which is the main receiving area for the input from cortical and subcortical brain regions. It mediates fear, anxiety, stress, and pain across species. Evidence suggests that the endocannabinoid system may be a promising target for modulating these processes. Cannabinoid and cannabinoid-related receptors have been identified in the amygdala of rodents, carnivores, and humans, but not in horses. This study aimed to investigate the gene expression of cannabinoid receptors 1 (CB1R) and 2 (CB2R), transient receptor potential vanilloid 1 (TRPV1), and peroxisome proliferator-activated receptor gamma (PPARγ) within the lateral nucleus of six equine amygdalae collected post mortem from an abattoir using quantitative real-time PCR, cellular distribution, and immunofluorescence. mRNA expression of CB1R and CB2R, but not TRPV1 or PPARγ, was detected. The percentage of immunoreactivity (IR) was calculated using ImageJ software. Cannabinoid receptor 1 immunoreactivity was absent in the somata but was strongly detected in the surrounding neuropil and varicosities and CB2R-IR was observed in the varicosities; TRPV1-IR showed moderate expression in the cytoplasm of somata and processes, while PPARγ-IR was weak-to-moderate in the neuronal nuclei. These findings demonstrate endocannabinoid system components in the equine amygdala and may support future studies on Cannabis spp. molecules acting on these receptors. Full article

Diacylglycerol-O-acyltransferase 1 (DGAT1, EC 2.3.1.20) is a pivotal enzyme in plant triacylglycerol (TAG) biosynthesis. Previous work identified conserved di-arginine (R) motifs (R-R, R-X-R, and R-X-X-R) in its N-terminal cytoplasmic acyl-CoA binding domain. To elucidate their functional significance, we engineered R-rich sequences in the N-termini of Tropaeolum majus and Zea mays DGAT1s. Comparative analysis with their respective non-mutant constructs showed that deleting or substituting R with glycine in the N-terminal region of DGAT1 markedly reduced lipid accumulation in both Camelina sativa seeds and Saccharomyces cerevisiae cells. Immunofluorescence imaging revealed co-localization of non-mutant and R-substituted DGAT1 with lipid droplets (LDs). However, disruption of an N-terminal di-R motif destabilizes DGAT1, alters LD organization, and impairs recombinant oleosin retention on LDs. Further evidence suggests that the di-R motif mediates DGAT1 retrieval from LDs to the endoplasmic reticulum (ER), implicating its role in dynamic LD–ER protein trafficking. These findings establish the conserved di-R motifs as important regulators of DGAT1 function and LD dynamics, offering insights for the engineering of oil content in diverse biological systems. Full article

Background: During infection with the cytomegalovirus (CMV), the membrane system of the infected cell is remodelled into a megastructure called the assembly compartment (AC). These extensive changes may involve the manipulation of the host cell proteome by targeting a pleiotropic function of the cell such as ubiquitination (Ub). In this study, we investigate whether the Ub system is required for the establishment and maintenance of the AC in murine CMV (MCMV)-infected cells Methods: NIH3T3 cells were infected with wild-type and recombinant MCMVs and the Ub system was inhibited with PYR-41. The expression of viral and host cell proteins was analyzed by Western blot. AC formation was monitored by immunofluorescence with confocal imaging and long-term live imaging as the dislocation of the Golgi and expansion of Rab10-positive tubular membranes (Rab10 TMs). A cell line with inducible expression of hemagglutinin (HA)-Ub was constructed to monitor ubiquitination. siRNA was used to deplete host cell factors. Infectious virion production was monitored using the plaque assay. Results: The Ub system is required for the establishment of the infection, progression of the replication cycle, viral gene expression and production of infectious virions. The Ub system also regulates the establishment and maintenance of the AC, including the expansion of Rab10 TMs. Increased ubiquitination of WASHC1, which is recruited to the machinery that drives the growth of Rab10 TMs, is consistent with Ub-dependent rheostatic control of membrane tubulation and the continued expansion of Rab10 TMs. Conclusions: The Ub system is intensively utilized at all stages of the MCMV replication cycle, including the reorganization of the membrane system into the AC. Disruption of rheostatic control of the membrane tubulation by ubiquitination and expansion of Rab10 TREs within the AC may contribute to the development of a sufficient amount of tubular membranes for virion envelopment. Full article

Background/Objective: Programmed cell death ligand-1 (PD-L1), which is overexpressed in certain tumors, inhibits the body’s natural immune response by providing an “off” signal that enables cancer cells to evade the immune system. It has been demonstrated that [¹⁷⁷Lu]Lu-DOTA-iPD-L1 (PD-L1 inhibitor cyclic peptide) promotes immune responses. This study aimed to synthesize and evaluate [¹⁸F]AlF-NOTA-iPD-L1 as a novel radiotracer for PD-L1 positron emission tomography (PET) imaging and as a potential theranostic pair for [¹⁷⁷Lu]Lu-DOTA-iPD-L1. Methods: The NOTA-iPD-L1 peptide conjugate was synthesized and characterized by U.V.-vis, I.R.-FT, and UPLC-mass spectroscopies. Radiolabeling was performed using [¹⁸F]AlF as the precursor, and the radiochemical purity (HPLC), partition coefficient, and serum stability were assessed. Cellular uptake and internalization (in 4T1 triple-negative breast cancer cells), binding competition, immunofluorescence, and Western blot assays were applied for the radiotracer in vitro characterization. Biodistribution in mice bearing 4T1 tumors was performed, and molecular imaging (Cerenkov images) of [¹⁸F]AlF-NOTA-iPD-L1 and [¹⁷⁷Lu]Lu-DOTA-iPD-L1 in the same mouse was obtained. Results: [¹⁸F]AlF-NOTA-iPD-L1 was prepared with a radiochemical purity greater than 97%, and it demonstrated high in vitro and in vivo stability, as well as specific recognition by the PD-L1 protein (IC₅₀ = 9.27 ± 2.69 nM). Biodistribution studies indicated a tumor uptake of 6.4% ± 0.9% ID/g at 1-hour post-administration, and Cerenkov images showed a high tumor uptake of both [¹⁸F]AlF-NOTA-iPD-L1 and ¹⁷⁷Lu-iPD-L1 in the same mouse. Conclusions: These results warrant further studies to evaluate the clinical usefulness of [¹⁸F]AlF-NOTA-iPD-L1/[¹⁷⁷Lu]Lu-DOTA-iPD-L1 as a radiotheranostic pair in combination with anti-PD-L1/PD1 immunotherapy. Full article

Thiophene derivatives are particularly attractive for application in drug development for their versatile pharmacological properties. We synthesized a series of four compounds with thiophene carboxamide as a scaffold. The structures were established based on HR-MS and 1D- and 2D-NMR. The purity of the compounds was established to be greater than 92% by thin-layer chromatography and NMR. The cytotoxic effects of the newly synthesized compounds were evaluated against the normal HaCaT cell line and A375, HT-29, and MCF-7 cancer cell lines. The cytotoxic assessment revealed that two compounds exhibit a significant cytotoxic effect on all cancer cell lines. To investigate their potential underlying mechanisms of action, several tests were performed: immunofluorescence imaging, caspase-3/7 assay, mitochondrial membrane potential (JC-1) assay, and 2′,7′–dichlorofluorescein diacetate (DCFDA) assay. MB-D2 proved to be the most cytotoxic and effective in terms of caspase 3/7 activation, mitochondrial depolarization and decrease in ROS production; these effects did not occur in normal HaCaT cells, revealing that MB-D2 has a high selectivity against A375 cancer cells. Full article

Background and Purpose: Glioblastoma remains a therapeutic challenge with a poor prognosis despite multimodal treatments. Reporter-based magnetic resonance imaging (MRI) offers a promising approach for tumor visualization, but its efficacy depends on sufficient reporter gene expression. This study aimed to develop a chimeric element-regulated ferritin heavy chain 1 (FTH1) reporter system to enhance MRI-based glioma detection while enabling targeted therapy via transferrin receptor (TfR)-mediated drug delivery. Methods: Using gene cloning techniques, we constructed a chimeric FTH1 expression system comprising tumor-specific PEG3 promoter (transcriptional control), bFGF-2 5′UTR (translational enhancement), and WPRE (mRNA stabilization). Lentiviral vectors delivered constructs to U251 glioblastoma cells and xenografts. FTH1/TfR expression was validated by Western blot and immunofluorescence. Iron accumulation was assessed via Prussian blue staining and TEM. MRI evaluated T2 signal changes. Transferrin-modified doxorubicin liposomes (Tf-LPD) were characterized for size and drug loading and tested for cellular uptake and cytotoxicity in vitro. In vivo therapeutic efficacy was assessed in nude mouse models through tumor volume measurement, MR imaging, and histopathology. Results: The chimeric system increased FTH1 expression significantly over PEG3-only controls (p < 0.01), with an increase of nearly 1.5-fold compared to the negative and blank groups and approximately a two-fold increase relative to the single promoter group, with corresponding TfR upregulation. Enhanced iron accumulation reduced T2 relaxation times significantly (p < 0.01), improving MR contrast. Tf-LPD (115 nm, 70% encapsulation) showed TfR-dependent uptake, inducing obvious apoptosis in high-TfR cells compared with that in controls. In vivo, Tf-LPD reduced tumor growth markedly in chimeric-system xenografts versus controls, with concurrent MR signal attenuation. Conclusions: The chimeric regulatory strategy overcomes limitations of single-element systems, demonstrating significant potential for integrated glioma theranostics. Its modular design may be adaptable to other reporter genes and malignancies. Full article

Integrating spatial transcriptomic data with immunofluorescence image data is challenging using existing tools due to their differences in spatial resolution. Immunofluorescence provides information about protein expression at the cellular or subcellular level, whereas spatial transcriptomic platforms typically rely on multicellular “spots” for RNA profiling. Our study coupled spatial transcriptomics of irradiated glioblastoma tissues with immunofluorescence for γH2AX, a marker of DNA damage within the nuclei of cells. We then compared gene expression in γH2AX-positive and negative regions within the tissue. There was significant interobserver variability in manual annotation of γH2AX positivity in multicellular spots by three different researchers (Kappa statistic = 0.345), despite all of them being familiar with γH2AX immunofluorescence and having predefined imaging parameters for annotation. This variability led to different researchers nominating different genes as being associated with DNA repair. To overcome this problem, we have developed a new tool using MATLAB. This tool performs “spot”-wise image analysis and uses researcher-defined parameters such as immunofluorescent marker intensity threshold and number of positive cells to annotate the “spots” as γH2AX positive or negative. The tissue with the most variability in manual annotation was annotated reproducibly by our MATLAB tool, leading to reproducible downstream analysis. Full article

Neuroinflammation, triggered by lipoxygenase (LOX), contributes to Alzheimer’s disease (AD) progression. Overexpression of LOX-5 in patients with AD serum highlights its role. This study assessed the efficacy of the LOX-inhibitor-peptide YWCS in an AD rat model induced by Aβ_25–35 injection. Cognitive tests, magnetic resonance imaging (MRI) scans, and molecular analyses were conducted. YWCS treatment significantly improved cognitive function, as evidenced by improved performance in the open field, novel object recognition, elevated plus maze, and Morris water maze tests. MRI scans revealed hippocampal shrinkage in AD rats and no changes were observed from YWCS treatment. Molecular analysis revealed altered expression of LOX-5, LOX-12, Aβ, γ-secretase components, p-Tau¹⁸¹, Akt, p-Akt, and p53 in AD rats. Immunofluorescence staining confirmed increased expression of LOX, Aβ, and p-Tau¹⁸¹ in the hippocampus of AD rats, which was reduced by YWCS treatment. Serum LOX levels were elevated in AD rats and significantly decreased after YWCS treatment, aligning with previous findings in human AD patients and AD cell models. YWCS offered improvements in behavioral and inflammatory marker regulation and also prevented progression of the disease, as shown by MRI results. These results suggest that YWCS, by targeting LOX, has the potential to be a promising therapeutic agent for AD. Full article

Aging is associated with altered itch perception, potentially due to changes in neuronal function and pruriceptive signaling. The underlying mechanisms, however, remain unclear. We investigated age-related differences in itch sensitivity at behavioral, cellular, and molecular levels. Young and old mice were intradermally injected with various pruritogens, including small molecules (histamine, chloroquine, and serotonin) and peptides (BAM8–22, AY-NH₂, and SLIGRL-NH₂). Scratching behavior and mechanical itch sensitivity were assessed, and calcium imaging was used to evaluate sensory neuron responses in the dorsal root ganglia. Additionally, immunofluorescence staining was performed to analyze the expression of TRPV1 and Cav3.2. Old mice exhibited reduced scratching behavior following injections, and their neuronal responses to histamine and chloroquine were diminished. Although all treated groups showed increased mechanical alloknesis, the effect was less pronounced in old animals. The expression of TRPV1 and Cav3.2 was also reduced in dorsal root ganglia neurons of old mice. These findings suggest that aging impairs both functional responsiveness and molecular signaling in sensory neurons, contributing to reduced chemical itch sensitivity in aged individuals. Full article

Background: Intervertebral disc degeneration (IVDD) is a major cause of chronic back pain. Recent studies suggest that ferroptosis, a form of cell death, contributes to the degeneration of nucleus pulposus cells (NPCs). This study explores a novel therapeutic strategy using cell-free fat extract (CEFFE), rich in cytokines, to mitigate IVDD by inhibiting oxidative stress-induced ferroptosis. Methods: In vitro, NPC degeneration was induced by TNF-α/TBHP. The effects of CEFFE on matrix metabolism were evaluated using Western blotting, RT-qPCR, and high-density culture, with regenerative effects measured via CCK-8 assays. Ferroptosis was assessed by Western blotting, immunofluorescence, and electron microscopy. In vivo, rats with caudal IVDD were treated with CEFFE for 4 weeks, and therapeutic efficacy was evaluated through imaging and histological analysis. Results: In vitro, CEFFE reduced TNF-α-induced inflammation and promoted matrix synthesis by inhibiting MAPK and NF-κB pathways. It also activated NRF2 to prevent TBHP-induced ferroptosis. In rats, CEFFE facilitated nucleus pulposus repair and significantly slowed disc degeneration. Conclusions: CEFFE is a promising strategy to delay IVDD progression by inhibiting ferroptosis, offering potential therapeutic benefits for disc degeneration. Full article

Inherited platelet disorders (IPDs) are rare bleeding disorders characterized by impaired platelet function and/or reduced blood platelet count. Their diagnosis typically relies on complex laboratory methods, including flow cytometry, aggregometry, and molecular genetic analysis. In recent years, immunofluorescence microscopy has been established as an alternative diagnostic method for IPDs. Background/Objectives: This study aims to validate a quantitative approach enhancing reproducibility through automated image analysis for diagnosing IPDs using immunofluorescence microscopy, with Bernard–Soulier Syndrome (BSS) and Glanzmann thrombasthenia (GT) as model IPDs. Methods: Native blood smears from patients with suspected BSS or GT were stained using a standardized immunofluorescence protocol targeting platelet surface glycoproteins, granules, and cytoskeletal components. The slides were analyzed using an automated fluorescence microscope, and a rule-based subpopulation analysis was implemented to quantify fluorescence signals. The results were compared to those of a healthy control group, as well as data from flow cytometry and molecular genetic testing. Results: The automated analysis successfully differentiated BSS and GT patients from healthy controls based on distinct fluorescence signal patterns. In BSS samples, CD42b (GPIbα) expression was absent or severely reduced, while GT samples showed a deficiency of CD41/CD61 (GPIIb/IIIa). The platelet size distribution confirmed macrothrombocytopenia in BSS patients. Flow cytometry and molecular genetic testing corroborated these findings, supporting the diagnostic reliability of the automated immunofluorescence microscopy approach. Conclusions: This proof-of-principle study demonstrates that automated quantitative immunofluorescence microscopy is a viable alternative for diagnosing IPDs, offering a standardized, objective, and efficient method, particularly in settings where flow cytometry is not feasible. Full article

Background/objectives: Brain cancer remains difficult to treat, with survival statistics stagnant for decades. The resistance of glioblastoma brain tumours can greatly challenge the effectiveness of conventional cancer radiotherapy. However, high dose rate radiotherapy has unique effects that allow for normal tissue sparing whilst maintaining tumour control. The addition of targeted radiosensitisers, such as the chemotherapeutic drug methotrexate (MTX) or the high-Z halogenated pyrimidine drug iododeoxyuridine (IUdR), can improve radiotherapy outcomes. Combining these radiosensitiser agents with ultra-high dose rate (UHDR) synchrotron X-rays can bear synergistic effects to enhance the efficacy of these multi-modal UHDR therapies, providing a means to overcome the radioresistance of brain cancer. Methods: Here, we use controlled in vitro assays following treatment, including a clonogenic assay to determine long-term cell survival and γH2AX immunofluorescent confocal microscopy to quantify double-strand DNA breaks (DSBs). Results: We find significant enhancement for highly synergistic combinations of IUdR+MTX with synchrotron X-rays. Cell survival results demonstrate 5.4 times increased 9L gliosarcoma cell killing when these agents are combined with UHDR synchrotron X-rays compared with conventional X-rays alone at the same 5 Gy dose. The underlying mechanisms are unveiled using γH2AX imaging and reveal significant increases in DSBs and dying cells following exposure to UHDR radiation. Conclusions: Our results demonstrate that highly synergistic combination treatments using UHDR synchrotron radiation can yield significantly improved brain cancer killing compared with conventional radiotherapy. We anticipate that these additive, multi-modal combination therapies will provide options for more targeted and effective use of radiotherapies for the future treatment of brain cancer. Full article

Background/Objectives: Our laboratory has been developing a Sindbis viral (SV) vector platform for treatments of several types of cancers. In this study, we assess treatment efficacy for metastatic and immunosuppressive pancreatic cancer. Methods: Orthotopic mouse models were generated by injection of tumor cells into the pancreatic parenchyma. Sindbis vectors were inoculated intraperitoneally. Imaging of tumors was performed by either MRI or in vivo imaging using luciferase. Flow cytometry, multi-immunofluorescence and elispot analysis were performed for certain tumors. Results: SV can infect and reduce pancreatic tumors in three mouse model systems: a model bearing human pancreatic tumors, a highly metastatic model, and a model that reflects the highly immunosuppressive, desmoplastic microenvironment common to human pancreatic cancer. Conclusions: Combination of SV vector expressing IL12 with an immune co-stimulatory agent, anti-OX40, can reduce tumors, facilitate an influx of immune response cells into the tumor microenvironment, and prevent tumors in mice rechallenged with tumor cells promising an effective treatment for pancreatic cancer. Full article

To overcome the limitations associated with chemically synthesized nanoparticles in cancer therapy, researchers have increasingly focused on developing nanoparticles with superior biocompatibility and prolonged tumor retention using biosynthetic methods. In this study, we first identified the presence of calcium peroxide nanoparticles (CaO₂ NPs) in the blood of individuals who had ingested calcium gluconate. Furthermore, the dropwise addition of calcium gluconate to human serum resulted in the spontaneous self-assembly of CaO₂ NPs. Next, following tail vein injection of fluorescently labeled CaO₂ NPs into subcutaneous tumor-bearing nude mice, we observed that the nanoparticles exhibited prolonged accumulation at the tumor sites compared to other organs through visible-light imaging. Immunofluorescence staining demonstrated that CaO₂ NPs co-localized with vesicular transport-associated proteins, such as PV-1 and Caveolin-1, as well as the albumin-binding-associated protein SPARC, suggesting that their transport from tumor blood vessels to the tumor site is mediated by Caveolin-1- and SPARC-dependent active transport pathways. Additionally, the analysis of various organs in normal mice injected with CaO₂ NPs at concentrations significantly higher than the experimental dose showed no apparent organ damage. Hemolysis assays indicated that hemolysis occurred only at calcium concentrations of 300 µg/mL, whereas the experimental concentration remained well below this threshold with no detectable hemolytic activity. In a subcutaneous tumor-bearing nude mouse model, treatment with docetaxel-loaded CaO₂ NPs showed a 68.5% reduction in tumor volume compared to free docetaxel (DTX) alone. These novel biosynthetic CaO₂ NPs demonstrated excellent biocompatibility, prolonged retention at the tumor site, safety, and drug-loading capability. Full article

The immunofluorescence assay is widely used for cellular biology and diagnosis applications. Such an antigen–antibody detection system enables the assessment and visualization of the expression and localization of target proteins. In the classical indirect immunofluorescence assay, secondary antibodies are conjugated to fluorophores. However, conventional secondary antibodies have limited applications due to their large size (150 kDa). Moreover, as animal-derived products, secondary antibodies are associated with ethical concerns and batch-to-batch variability. In this study, we developed fluorescence-labeled recombinant nanobodies as secondary antibodies by utilizing previously established anti–mouse and anti–rabbit IgG secondary nanobodies in combination with the self-labeling SNAP-tag. Nanobodies, which are significantly smaller (15 kDa), are capable to detect primary antibodies produced in mice and rabbits. The SNAP-tag (20 kDa) enables site-specific binding of various O⁶-benzylguanine (BG)-modified fluorophores to the recombinant nanobodies. These recombinant nanobodies were produced using mammalian cell expression system, and their specific binding to mouse or rabbit antibodies was validated using flow cytometry and multi-color fluorescence microscopy. The low cost, easy of expression, purification and site-specific conjugation procedures for these anti–mouse and anti–rabbit IgG secondary nanobodies make them an attractive alternative to traditional secondary antibodies for indirect immunofluorescence assays. Full article