Changes In Cellular Morphology Research Articles

Stable Isotope Probing-nanoFTIR for Quantitation of Cellular Metabolism and Observation of Growth-Dependent Spectral Features.

This study utilizes nanoscale Fourier transform infrared spectroscopy (nanoFTIR) to perform stable isotope probing (SIP) on individual bacteria cells cultured in the presence of 13C-labelled glucose. SIP-nanoFTIR simultaneously quantifies single-cell metabolism through infrared spectroscopy and acquires cellular morphological information via atomic force microscopy. The redshift of the amide I peak corresponds to the isotopic enrichment of newly synthesized proteins. These observations of single-cell translational activity are comparable to those of conventional methods, examining bulk cell numbers. Observing cells cultured under conditions of limited carbon, SIP- nanoFTIR is used to identify environmentally-induced changes in metabolic heterogeneity and cellular morphology. Individuals outcompeting their neighboring cells will likely play a disproportionately large role in shaping population dynamics during adverse conditions or environmental fluctuations. Additionally, SIP-nanoFTIR enables the spectroscopic differentiation of specific cellular growth phases. During cellular replication, subcellular isotope distribution becomes more homogenous, which is reflected in the spectroscopic features dependent on the extent of 13C-13C mode coupling or to specific isotopic symmetries within protein secondary structures. As SIP-nanoFTIR captures single-cell metabolism, environmentally-induced cellular processes, and subcellular isotope localization, this technique offers widespread applications across a variety of disciplines including microbial ecology, biophysics, biopharmaceuticals, medicinal science, and cancer research.

Neuronal microstructural changes in the human brain are associated with neurocognitive aging.

Gray matter (GM) alterations play a role in aging-related disorders like Alzheimer's disease and related dementias, yet MRI studies mainly focus on macroscopic changes. Although reliable indicators of atrophy, morphological metrics like cortical thickness lack the sensitivity to detect early changes preceding visible atrophy. Our study aimed at exploring the potential of diffusion MRI in unveiling sensitive markers of cortical and subcortical age-related microstructural changes and assessing their associations with cognitive and behavioral deficits. We leveraged the Human Connectome Project-Aging cohort that included 707 participants (394 female; median age = 58, range = 36-90 years) and applied the powerful mean apparent diffusion propagator model to measure microstructural parameters, along with comprehensive behavioral and cognitive test scores. Both macro- and microstructural GM characteristics were strongly associated with age, with widespread significant microstructural correlations reflective of cellular morphological changes, reduced cellular density, increased extracellular volume, and increased membrane permeability. Importantly, when correlating MRI and cognitive test scores, our findings revealed no link between macrostructural volumetric changes and neurobehavioral performance. However, we found that cellular and extracellular alterations in cortical and subcortical GM regions were associated with neurobehavioral performance. Based on these findings, it is hypothesized that increased microstructural heterogeneity and decreased neurite orientation dispersion precede macrostructural changes, and that they play an important role in subsequent cognitive decline. These alterations are suggested to be early markers of neurocognitive performance that may distinctly aid in identifying the mechanisms underlying phenotypic aging and subsequent age-related functional decline.

Lung cancer cell-intrinsic IL-15 promotes cell migration and sensitizes murine lung tumors to anti-PD-L1 therapy

BackgroundIL-15 plays a vital role in enhancing NK cell- and T-cell-mediated antitumor immune responses; however, the direct effect of IL-15 on tumor cells has not been fully elucidated. Herein, we investigated the effect of IL-15 on lung adenocarcinoma cells.MethodsSilencing and overexpression techniques were used to modify endogenous IL-15 expression in tumor cells. Transwell assays were used to assess tumor cell migration and invasion; a live-cell analysis system was used to evaluate cell motility; cellular morphological changes were quantified by confocal fluorescence microscopy; the molecular mechanisms underlying the effect of IL-15 on tumor cells were analyzed by western blotting; and RhoA and Cdc42 activities were evaluated by a pulldown assay. NCG and C57BL/6 mouse models were used to evaluate the functions of IL-15 in vivo.ResultsCancer cell-intrinsic IL-15 promoted cell motility and migration in vitro and metastasis in vivo via activation of the AKT-mTORC1 pathway; however, exogenous IL-15 inhibited cell motility and migration via suppression of the RhoA-MLC2 axis. Mechanistic analysis revealed that both the intracellular and extracellular IL-15-mediated effects required the expression of IL-15Rα by tumor cells. Detailed analyses revealed that the IL-2/IL-15Rβ and IL-2Rγ chains were undetected in the complex formed by intracellular IL-15 and IL-15Rα. However, when exogenous IL-15 engaged tumor cells, a complex containing the IL-15Rα, IL-2/IL-15Rβ, and IL-2Rγ chains was formed, indicating that the differential actions of intracellular and extracellular IL-15 on tumor cells might be caused by their distinctive modes of IL-15 receptor engagement. Using a Lewis lung carcinoma (LLC) metastasis model, we showed that although IL-15 overexpression facilitated the lung metastasis of LLC cells, IL-15-overexpressing LLC tumors were more sensitive to anti-PD-L1 therapy than were IL-15-wild-type LLC tumors via an enhanced antitumor immune response, as evidenced by their increased CD8+ T-cell infiltration compared to that of their counterparts.ConclusionsCancer cell-intrinsic IL-15 and exogenous IL-15 differentially regulate cell motility and migration. Thus, cancer cell-intrinsic IL-15 acts as a double-edged sword in tumor progression. Additionally, high levels of IL-15 expressed by tumor cells might improve the responsiveness of tumors to immunotherapies.

Abstract P70: HIGH-THROUGHPUT IMAGING-BASED PHENOTYPIC PROFILING OF IMMUNE CELLS FROM BRCA2 VARIANT CARRIERS AND NON-CARRIERS

Abstract Mutated germline alleles in the DNA damage repair (DDR) genes BRCA1/2 are major risk factors for breast and ovarian cancers. Recent studies have found that BRCA1-mutant tumors tend to have higher abundance of immune cells within the tumor microenvironment, but much better response was observed from the BRCA2-mutant tumors when given immune-checkpoint inhibitors, suggesting possible functional and phenotypic effects of germline BRCA2 mutations on the immune cells. However, most existing studies focus on investigating the genomic instability and phenotypes of hereditary mutations in breast and ovarian cancer cells. Thus, the impact of these mutations on immune cells’ phenotypes and functions remains largely unknown. Here, we report a study of immune cells with and without BRCA2 mutations using High-throughput Imaging-based Phenotypic Profiling (HIPP), which can identify changes in cellular morphology and protein subcellular localization patterns that are not obvious from other traditional functional assays. We performed HIPP on immune cells from nine BRCA2 pathogenic variant carriers and eight non-carriers. Automatic quantification of ~200 phenotypic features from each single cell revealed that CD8+ T cells from BRCA2 variant carriers have distinct and quantifiable changes in their morphological features (such as cytoskeleton structures) and immunological features (such as Granzyme B expressions). Moreover, we found that BRCA2-mutant CD8+ T cells proliferate faster than normal CD8+ T cells. Our results suggest that BRCA2 mutations affect the functions and phenotypes of immune cells. Some of these phenotypic features and markers may be used to classify BRCA2 variants and predict the pathogenicity of variants of uncertain significance (VUS). Our study provides new insight into the immune phenotyping of cancer patients at individual level and may help to stratify patients, leading to the development of more effective therapeutic strategies. This method can potentially extend to other germline variants and overcome the challenge of VUS interpretation in clinical management. Citation Format: Guorui Zhong, Oscar Fu, Claresta Yeo, Siao Ting Chong, Sock Hoai Chan, Joanne Ngeow, Lit-Hsin Loo. HIGH-THROUGHPUT IMAGING-BASED PHENOTYPIC PROFILING OF IMMUNE CELLS FROM BRCA2 VARIANT CARRIERS AND NON-CARRIERS [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P70.

Harnessing Microbial Effectors for Macrophage-Mediated Drug Delivery.

Macrophage-based drug delivery systems are promising, but their development is still in its infancy, with many limitations remaining to be addressed. Our aim was to design a system harnessing microbial effectors to facilitate controlled drug cargo expulsion from macrophages to enable the use of more toxic drugs without adding to the risk of off-target detrimental effects. The pore forming and actin polymerizing Listeria monocytogenes effectors listeriolysin-O (LLO) and actin assembly-inducing protein (ActA) were synthesized using a novel green fluorescent protein (GFP)-linked heterologous expression system. These effectors were coated onto polystyrene beads to generate "synthetic cargo" before loading into primary M1 macrophages. Bead uptake and release from macrophages were evaluated by using high-throughput quantitative imaging flow cytometry and confocal microscopy. In vitro results confirmed appropriate activity of synthesized effectors. Coating of these effector proteins onto polystyrene beads (simulated drug cargo) resulted in changes in cellular morphology, bead content, and intracellular bead localization, which may support an interpretation of the induced release of these beads from the cells. This forms the basis for further investigation to fully elucidate any potential release mechanisms. Bacterial effectors ActA and LLO successfully effectuated actin polarization and protrusions from cell membranes similar to those seen in cells infected with Listeria spp., illustrating the potential of using these effectors and production methods for the development of an endogenous drug delivery system capable of low-risk, targeted release of high potency drugs.

Phenotypic comparison and the potential antitumor function of immortalized bone marrow-derived macrophages (iBMDMs).

Macrophages are an important component of innate immunity and involved in the immune regulation of multiple diseases. The functional diversity and plasticity make macrophages to exhibit different polarization phenotypes after different stimuli. During tumor progression, the M2-like polarized tumor-associated macrophages (TAMs) promote tumor progression by assisting immune escape, facilitating tumor cell metastasis, and switching tumor angiogenesis. Our previous studies demonstrated that functional remodeling of TAMs through engineered-modifying or gene-editing provides the potential immunotherapy for tumor. However, lack of proliferation capacity and maintained immune memory of infused macrophages restricts the application of macrophage-based therapeutic strategies in the repressive tumor immune microenvironment (TIME). Although J2 retrovirus infection enabled immortalization of bone marrow-derived macrophages (iBMDMs) and facilitated the mechanisms exploration and application, little is known about the phenotypic and functional differences among multi kinds of macrophages. HE staining was used to detect the biosafety of iBMDMs, and real-time quantitative PCR, immunofluorescence staining, and ELISA were used to detect the polarization response and expression of chemokines in iBMDMs. Flow cytometry, scratch assay, real-time quantitative PCR, and crystal violet staining were used to analyze its phagocytic function, as well as its impact on tumor cell migration, proliferation, and apoptosis. Not only that, the inhibitory effect of iBMDMs on tumor growth was detected through subcutaneous tumor loading, while the tumor tissue was paraffin sectioned and flow cytometry was used to detect its impact on the tumor microenvironment. In this study, we demonstrated iBMDMs exhibited the features of rapid proliferation and long-term survival. We also compared iBMDMs with RAW264.7 cell line and mouse primary BMDMs with in vitro and in vivo experiments, indicating that the iBMDMs could undergo the same polarization response as normal macrophages with no obvious cellular morphology changes after polarization. What's more, iBMDMs owned stronger phagocytosis and pro-apoptosis functions on tumor cells. In addition, M1-polarized iBMDMs could maintain the anti-tumor phenotypes and domesticated the recruited macrophages of receptor mice, which further improved the TIME and repressed tumor growth. iBMDMs can serve as a good object for the function and mechanism study of macrophages and the optional source of macrophage immunotherapy.

A physics-informed neural network framework to investigate nonlinear and heterogenous shrinkage of drying plant cells

This paper introduces a novel Physics-Informed Neural Network-based (PINN-based) multi-domain computational framework to analyse nonlinear and heterogeneous morphological variations of plant cells during drying. Here, two distinct models are involved: PINN-MT to simulate mass transfer; and PINNNS to simulate nonlinear shrinkage. The models are coupled to examine cellular morphological changes resulting from moisture loss during drying. Firstly, the coupled framework, in tandem with homogeneous conditions, operates in parallel, allowing the mutual parameters to update between models. This approach demonstrates ability to approximate homogeneous cellular shrinkage within a tissue, factoring in the influence of surrounding plant cells. Secondly, non-uniform cell wall properties and heterogeneous boundary conditions are incorporated into this computational framework through domain decomposition. Inherent capabilities of neural networks allow for seamless integration of multiple domains, with additional loss terms introduced at interfaces. The framework shows capacity to account for drastic and non-uniform morphological variations of plant cells even under extreme drying conditions, which is the key novelty and has been a challenging task for existing traditional computational methods. Hence, the proposed computational approach offers an innovative avenue for understanding nonlinear and heterogeneous morphological variations not only for plant cells, but also for soft matter in general.

Melatonin prevents pulmonary fibrosis caused by PM2.5 exposure by targeting epithelial-mesenchymal transition

Pulmonary fibrosis is a lung disorder characterized by the accumulation of abnormal extracellular matrix, scar tissue formation, and tissue stiffness. Type II alveolar epithelial cells (AEII) play a critical role in repairing lung tissue after injury, and repeated injury to these cells is a key factor in the development of pulmonary fibrosis. Chronic exposure to PM2.5, a type of air pollution, has been shown to increase the incidence and severity of pulmonary fibrosis by enhancing the activation of EMT in lung epithelial cells. Melatonin, a hormone with antioxidant properties, has been shown to prevent EMT and reduce fibrosis in previous studies. However, the mechanism through which melatonin targets EMT to prevent pulmonary fibrosis caused by PM2.5 exposure has not been extensively discussed before. In this current study, we found that melatonin effectively prevented pulmonary fibrosis caused by prolonged exposure to PM2.5 by targeting EMT. The study demonstrated changes in cellular morphology and expression of EMT markers. Furthermore, the cell migratory potential induced by prolonged exposure to PM2.5 was greatly reduced by melatonin treatment. Finally, in vivo animal studies showed reduced EMT markers and improved pulmonary function. These findings suggest that melatonin has potential clinical use for the prevention of pulmonary fibrosis.

BRD4L cooperates with MYC to block local tumor invasion via suppression of S100A10

Targeted therapy based on BRD4 and MYC shows promise due to their well-researched oncogenic functions in cancer, but their tumor-suppressive roles are less understood. In this study, we employ a systematic approach to delete exons that encode the low-complexity domain (LCD) of BRD4L in cells by using CRISPR–Cas9. In particular, the deletion of exon 14 (BRD4–E14) results in cellular morphological changes towards spindle-shaped and loosely packed. BRD4–E14 deficient cells show increased cell migration and reduced cell adhesion. The expression of S100A10 was significantly increased in cells lacking E14. BRD4L binds with MYC via the E14-encoded region of the LCD to inhibit the expression of S100A10. In cancer tissues, there is a positive correlation between BRD4 and MYC, while both of these proteins are negatively associated with S100A10 expression. Finally, knocking out the BRD4–E14 region or MYC promotes tumor growth in vivo. Together, these data support a tumor-suppressive role of BRD4L and MYC in some contexts. This discovery emphasizes the significance of a discreetly design and precise patient recruitment in clinical trials that testing cancer therapy based BRD4 and MYC.

Abstract 582: Oncogenic PIK3CA fortifies immune determinants in vascular cancers

Abstract Angiosarcomas are a group of vascular cancers that form malignant blood vessels. These malignancies are seemingly inflamed primarily due to their pathognomonic nature, which consists of irregular endothelium and tortuous blood channels. The malignant vascular spaces host a variety of blood and immune components, where a controlled scavenging system is necessary for tumor maintenance. Oncogenes may convey molecular signals to foster the tumor immune niche, and PIK3CA mutations that induce continuous activation of the PI3K pathway occur in a subset of angiosarcomas. Despite the undoubtedly oncogenic properties of PIK3CA mutations, the mechanistic role of these mutations - particularly the potential for controlling the hemato-endothelial system in angiosarcomas - remains undetermined. In this study, we demonstrate the functional consequences of oncogenic PIK3CA and its molecular properties in angiosarcoma cells. We created isogenic cell clones from two angiosarcoma cell lines engineered to harbor hotspot mutations in PIK3CA using CRISPR/Cas9: two homozygous PIK3CA H1047R cell clones (C8 and C35) and one wild-type (WT) clone were generated from DHSA-1426 angiosarcoma cells; and one homozygous mutant clone was created from COSB cells. We found that PIK3CA mutant cells constitutively activated the PI3K/AKT/mTOR pathway at a greater level than that of WT cells. Bulk- and single cell(sc)-RNA-seq data revealed that mutant cells enriched gene sets associated with PI3K signaling and other oncogenic pathways including KRAS, MAPK, and TGF-beta. Also, mutant cells significantly increased cytokines regulating immune cell migration and chemotaxis such as IL-8 and MCP-1. In addition to the common features, we identified functional and molecular divergence between mutant clones. Noticeable changes in cellular morphology, growth kinetics, and tube-forming capacity were observed between two mutant clones. C35 mutant cells were more sensitive to both pan-PI3K and PI3K-alpha specific inhibitor, and DNA damage was greater than in the C8 clone. C35 cells enhanced activation of IL-6/STAT3 signaling compared to C8, despite the increment of the aforementioned immune cytokines in both mutants. Moreover, our comprehensive analysis of bulk- and sc-RNA-seq, ATAC-seq, and metabolomics data identified molecular signatures associated with metabolic vulnerabilities of C35 cells. In summary, we demonstrate that oncogenic PIK3CA perpetuates PI3K signaling activation and reinforces a supply for immune signaling. Furthermore, our data suggest that PIK3CA mutations contribute to establishing cellular heterogeneity that reveals a spectrum of vascular, immunogenic, and metabolic activities. Our ongoing work involves developing an experimental model of angiosarcoma using induced pluripotent stem cells to examine if oncogenic PIK3CA controls immune determinants during malignant endothelial transformation. Citation Format: Donghee Lee, Emma Kozurek, Md Abdullah, Rong Li, Erin B. Dickerson, Jong Hyuk Kim. Oncogenic PIK3CA fortifies immune determinants in vascular cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 582.

Abstract 1430: Development of transparent transgenic zebrafish model to study anti-cancer drug induced in vivo cardiotoxicity

Abstract Cardiotoxicity is the second leading cause of death among cancer survivors and can manifest as arrhythmia, acute coronary syndrome, acute heart failure, or pericarditis/myocarditis. Chronic myocyte damage and apoptosis can transition from an asymptomatic phase to adaptive myocardial hypertrophy, followed by decreased left ventricular ejection fraction, and eventual heart failure. Cardio-oncology is an emerging field that studies cancer therapy-associated cardiotoxicities. The development of reliable and effective preclinical models for cardiotoxicity prediction and prevention is essential to improving the long-term cardiac health of cancer patients. Zebrafish are a promising animal model for the field of cardio-oncology and have emerged as an invaluable tool for investigating genetic and molecular aspects of cardiovascular research. Zebrafish heart cells are like human heart cells at the molecular level, making them an ideal system for studying the genetic underpinning of cardiac function. Zebrafish myl7 gene, a myosin light chain-7 gene, is a regulatory gene of heart orthologs to human MYL7. In-vivo imaging is limited due to the opacification of skin and subdermal structures. To conduct in-vivo whole organism imaging studies, skin transparency is a primary requirement, necessitating the blocking of pigmentation. The Zebrafish Casper mutant maintains skin transparency throughout its life, offering the ideal combination of sensitivity and resolution for in-vivo analyses and imaging. In this study, we have developed a novel transparent transgenic zebrafish model and established time-lapse in-vivo confocal microscopy to study cellular phenotype/pathologies of cardiomyocytes. The newly developed homozygous transparent transgenic Casper/myl7:RFP; annexin-5:YFP/NF-kB: GFP/mpeg1:mcherryFP, allows us to study cell death process (Annexin5) and inflammatory activity (NF-kB) in microglia and cardiomyocytes. We have successfully established in vivo fluorescence and confocal microscopy in this newly developed strain, enabling the study of cellular morphology and function changes under various conditions. This will allow us to elucidate cell death patterns, and cardio-inflammatory and cardiotoxicity pathways. Citation Format: Surendra K. Rajpurohit, Taitum Gossman, Sai Nasanally, Keshu Bhatt, Aiden Van Derhei, Simran Hotwani, Devan-Anmol Manhiani, Rohan Pasi, Jiaqi Mi, Vishal Arora, Balakrishna Lokeshwar. Development of transparent transgenic zebrafish model to study anti-cancer drug induced in vivo cardiotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1430.

Terahertz Radiation Modulates Neuronal Morphology and Dynamics Properties.

Terahertz radiation falls within the spectrum of hydrogen bonding, molecular rotation, and vibration, as well as van der Waals forces, indicating that many biological macromolecules exhibit a strong absorption and resonance in this frequency band. Research has shown that the terahertz radiation of specific frequencies and energies can mediate changes in cellular morphology and function by exciting nonlinear resonance effects in proteins. However, current studies have mainly focused on the cellular level and lack systematic studies on multiple levels. Moreover, the mechanism and law of interaction between terahertz radiation and neurons are still unclear. Therefore, this paper analyzes the mechanisms by which terahertz radiation modulates the nervous system, and it analyzes and discusses the methods by which terahertz radiation modulates neurons. In addition, this paper reviews the laws of terahertz radiation's influence on neuronal morphology and kinetic properties and discusses them in detail in terms of terahertz radiation frequency, energy, and time. In the future, the safety of the terahertz radiation system should be considered first to construct the safety criterion of terahertz modulation, and the spatial resolution of the terahertz radiation system should be improved. In addition, the systematic improvement of the laws and mechanisms of terahertz modulation of the nervous system on multiple levels is the key to applying terahertz waves to neuroscience. This paper can provide a platform for researchers to understand the mechanism of the terahertz-nervous system interaction, its current status, and future research directions.

3-bromopyruvate induces morphological alteration and may initiate programmed cell death in Cryptococcus neoformans cells.

3-Bromopyruvate (3BP), known for its potent anticancer properties, also exhibits remarkable efficacy against the pathogenic fungus Cryptococcus neoformans. So far it has been proven that the main fungicidal activity of 3BP is based on ATP depletion and a reduction of intracellular level of glutathione. The presented study includes a broad range of methods to further investigate the mechanistic effects of 3BP on C. neoformans cells. The use of flow cytometry allowed a thorough examination of their survival during 3BP treatment, while observations using electron microscopy made it possible to note the changes in cellular morphology. Utilizing ruthenium red, the study suggests a mitochondrial pathway may initiate programmed cell death in response to 3BP. Analysis of free radical generation and gene expression changes supports this hypothesis. These findings enhance comprehension of 3BP's mechanisms in fungal cells, paving the way for its potential application as a therapeutic agent against cryptococcosis.

Palmitate Compromises C6 Astrocytic Cell Viability and Mitochondrial Function.

Consumption of high-fat diets (HFD) is associated with brain alterations, including changes in feeding behavior, cognitive decline, and dementia. Astrocytes play a role in HFD-induced neuroinflammation and brain dysfunction; however, this process is not entirely understood. We hypothesized that exposure to saturated fatty acids can compromise astrocyte viability and mitochondrial function. The C6 (astrocytes) cell line was treated with palmitate or stearate (200 µM and 400 µM) for 6 h. Cell viability, morphology, inflammatory markers, and oxidative stress were evaluated. To assess mitochondrial function, various parameters were measured (membrane potential, mass, respiration, and complex activities). We observed that 6 h of treatment with 400 µM palmitate decreased cell viability, and treatment with 200 µM palmitate changed the astrocyte morphology. Palmitate increased inflammatory markers (TNF-α and IL6) but did not induce oxidative stress. Palmitate significantly decreased the mitochondrial membrane potential and mitochondrial mass. Complex I activity also decreased in palmitate-treated cells; however, no changes were observed in mitochondrial respiration. In conclusion, palmitate, a saturated fatty acid, induces inflammation and impairs mitochondrial function, leading to reduced astrocytic cell viability and changes in cellular morphology. Our study provides valuable insights into the potential mechanisms underlying the relationship between saturated fatty acids, astrocytes, and mitochondrial function in obesity-related brain dysfunction.

Mild hypothermia reduces lipopolysaccharide-induced microglial activation via down-regulation of Tent5c

Microglial activation is a critical factor in the pathogenesis and progression of neuroinflammatory diseases. Mild hypothermia, known for its neuroprotective properties, has been shown to alleviate microglial activation. In this study, we explore the differentially expressed (DE) mRNAs and long non-coding RNAs (lncRNAs) in BV-2 microglial cells under different conditions: normal temperature (CN), mild hypothermia (YT), normal temperature with lipopolysaccharide (LPS), and mild hypothermia with LPS (LPS + YT). Venn analysis revealed 119 DE mRNAs that were down-regulated in the LPS + YT vs LPS comparison but up-regulated in the CN vs LPS comparison, primarily enriched in Gene Ontology terms related to immune and inflammatory responses. Furthermore, through Venn analysis of YT vs CN and LPS + YT vs LPS comparisons, we identified 178 DE mRNAs and 432 DE lncRNAs. Among these transcripts, we validated the expression of Tent5c at the protein and mRNA levels. Additionally, siRNA-knockdown of Tent5c attenuated the expression of pro-inflammatory genes (TNF-α, IL-1β, Agrn, and Fpr2), cellular morphological changes, NLRP3 and p-P65 protein levels, immunofluorescence staining of p-P65 and number of cells with ASC-speck induced by LPS. Furthermore, Tent5c overexpression further potentiated the aforementioned indicators in the context of mild hypothermia with LPS treatment. Collectively, our findings highlight the significant role of Tent5c down-regulation in mediating the anti-inflammatory effects of mild hypothermia.

Comparative analysis of process intensification technologies (PIT) for improved cell disruption and lipid recovery in Aurantiochytrium sp. SW1 microalgae

SummaryThis study explores enhancing lipid recovery from the lipid‐rich marine microalgae Aurantiochytrium sp. SW1, known for its robust cell wall posing challenges, which necessitates energy‐intensive cell‐disruption for efficient intracellular lipid extraction. Despite advancements in other microalgae, there is lack of reports on efficient cell disruption for Aurantiochytrium sp. SW1 which is vital to reduce the energy costs and minimising the downstream process while maximising lipid yields. Three process intensification technologies (PIT) – microwave treatment, autoclave treatment and ultrasonication were employed on wet Aurantiochytrium sp. SW1 cells to improve the lipid extraction efficiency. Assessing the disruption effectiveness via lipid content, suspension turbidity and particle size distribution, alongside scanning electron microscopy (SEM) for cellular morphology changes post‐PIT treatments, this study indicates all three methods reduced particle size with prolonged treatment time, implying successful cell wall disruption and intracellular component release. Ultrasonication, especially at 50 W power for 10 min, exhibited the highest efficacy, yielding lipid content of 67.76 ± 0.03%. Microscopic analysis unveiled ultrasonication‐induced cell shrinkage and increased membrane permeability, supporting its effectiveness in enhancing solvent penetration for improved lipid recovery. This research underscores PIT techniques' potential as sustainable, energy‐efficient strategies for lipid extraction from Aurantiochytrium sp. SW1 microalgae.

Protective Effects of Lycium barbarum Polysaccharides, Mulberry Leaf Flavonoids, and Ginkgo Leaf Flavonoids on HBZY-1 Cell Injury: An Investigation of Lactate Dehydrogenase and Total Nitric Oxide Synthase

The impacts of natural plant extracts, including Lycium barbarum polysaccharides (LBP), mulberry leaf flavonoids (MLF), and ginkgo leaf flavonoids (GLF), on high glucose (HG)-induced injury in HBZY-1 mesangial cells were analyzed. LBP, MLF, and GLF were individually extracted, and their purities were determined. HBZY-1 cells cultured in low glucose (LG) served as the Normal group (NG), while HG culture represented the HG group. Subsequently, HBZY-1 cells in the HG group were treated with 300 μg/mL of LBP, MLF, and GLF, resulting in the HG+LBP, HG+MLF, and HG+GLF groups, respectively. Cell viability (CV) was assessed using the MTT assay, and cellular morphological changes were observed through HE staining. Meanwhile, lactate dehydrogenase (LDH) and nitric oxide (NO) levels were examined using the microplate method. Furthermore, malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) levels were detected by ELISA. mRNA levels in IL-6, IL-1β, and TNF-α were assessed using real-time quantitative polymerase chain reaction (rt-qPCR), and Collagen IV was examined using immunofluorescence. The results revealed that the polysaccharide content in the LBP extract was 63.57%, the flavonoid contents in the MLF and GLF extracts were 55.40% and 48.62%, respectively. Based on the NG group, HG group exhibited decreased CV, increased LDH and MDA, decreased NO, SOD, and GSH-Px, elevated mRNA levels in IL-6, IL-1β, and TNF-α, and reduced Collagen IV, showing great differences (P < 0.05). Based on the HG group, the HG+LBP, HG+MLF, and HG+GLF groups demonstrated increased CV, decreased LDH and MDA levels, increased NO, SOD, and GSH-Px levels, decreased mRNA levels in IL-6, IL-1β, and TNF-α, and increased Collagen IV, presenting obvious differences (P < 0.05). Comparing to the HG+LBP group, the HG+MLF and HG+GLF groups exhibited enhanced CV, downshifted LDH and MDA, elevated NO, SOD, and GSH-Px, downregulated mRNA in IL-6, IL-1β, and TNF-α, and increased Collagen IV (P < 0.05). No significant changes in CV, LDH, MDA, NO, SOD, GSH-Px, IL-6, IL-1β, TNF-α, or Collagen IV were observed in the HG+MLF and HG+GLF groups to the NG group (P >0.05). In conclusion, natural plant extracts such as LBP, MLF, and GLF exerted protective effects against injury in HBZY-1 mesangial cells caused by HG and possessed significant anti-inflammatory, antioxidant stress, and antifibrotic damage properties.

Neuroprotective effects of polyphyllin VI against rotenone-induced toxicity in SH-SY5Y cells

BackgroundA substantial body of evidence is drawing connections between Parkinson's disease (PD) and the phenomena of oxidative stress and mitochondrial dysfunction. Polyphyllin VI (PPVI), an active compound found in Rhizoma Paridis-commonly known as Chonglou (CL) in China, has been identified for its various pharmacological properties, including anti-tumor and anti-inflammatory effects. ObjectiveIn the present study, an in vitro model of PD was established by treating SH-SY5Y cells with rotenone (ROT), to evaluate the potential neuroprotective effects of polyphyllin VI and its underlying mechanism. MethodsSH-SY5Y cells were treated with ROT to establish an in vitro model of PD. The effects of polyphyllin VI on cell viability were assessed using the resazurin assay. Cell morphology was examined using a microscope. The YO-PRO-1/PI was used to detect apoptosis. Mito-Tracker Red CMXRos, Mito-Tracker Green, and JC-1 were used to detect the effects of polyphyllin Ⅵ on mitochondrial viability, morphology, and function. Oxidative stress-related marker detection kits were used to identify the effects of polyphyllin VI on oxidative stress. Western blot analysis was employed to investigate the signaling pathways associated with neuroprotection. ResultsPPVI increased ROT-induced SH-SY5Y cell viability and improved ROT-induced cellular morphological changes. PPVI ameliorated ROT-induced oxidative stress status, and attenuated mitochondrial function and morphological changes. PPVI may exert neuroprotective effects through FOXO3α/CREB1/DJ-1-related signaling pathways. ConclusionThese preliminary findings suggested that PPVI possesses neuroprotective attributes in vitro, and it may be a potential candidate for PD treatment. However, extensive research is necessary to fully understand the mechanisms of PPVI and its effectiveness both in vitro and in vivo.

Comparative genomics incorporating translocation renal cell carcinoma mouse model reveals molecular mechanisms of tumorigenesis.

Translocation renal cell carcinoma (tRCC) most commonly involves an ASPSCR1-TFE3 fusion, but molecular mechanisms remain elusive and animal models are lacking. Here, we show that human ASPSCR1-TFE3 driven by Pax8-Cre (a credentialed clear cell RCC driver) disrupted nephrogenesis and glomerular development, causing neonatal death, while the clear cell RCC failed driver, Sglt2-Cre, induced aggressive tRCC (as well as alveolar soft part sarcoma) with complete penetrance and short latency. However, in both contexts, ASPSCR1-TFE3 led to characteristic morphological cellular changes, loss of epithelial markers, and an epithelial-mesenchymal transition. Electron microscopy of tRCC tumors showed lysosome expansion, and functional studies revealed simultaneous activation of autophagy and mTORC1 pathways. Comparative genomic analyses encompassing an institutional human tRCC cohort (including a hitherto unreported SFPQ-TFEB fusion) and a variety of tumorgraft models (ASPSCR1-TFE3, PRCC-TFE3, SFPQ-TFE3, RBM10-TFE3, and MALAT1-TFEB) disclosed significant convergence in canonical pathways (cell cycle, lysosome, and mTORC1) and less established pathways such as Myc, E2F, and inflammation (IL-6/JAK/STAT3, interferon-γ, TLR signaling, systemic lupus, etc.). Therapeutic trials (adjusted for human drug exposures) showed antitumor activity of cabozantinib. Overall, this study provides insight into MiT/TFE-driven tumorigenesis, including the cell of origin, and characterizes diverse mouse models available for research.

Anticancer potential of toxin extracts from vital organs of milk spotted puffer fish Chelonodon patoca on human cancer cell lines

Among aquatic creatures, fish are the most diversified organisms that have emerged among other vertebrates. The objective of this research was to evaluate the anticancer properties of toxin extracts from the marine puffer fish Chelonodon patoca. The toxins were extracted from the samples by homogenizing with acetic acid, and the clear supernatant was subjected to anticancer activities by MTT for cytotoxicity and cell viability on both Vero and cancer cell lines (A-549, AGS, HT-29, and MDA-MB-231). More than 70% of the normal cells were viable up to 200 µg/ml of toxin extracts so that hence the concentration was fixed below 200 µg/ml for the cell viability study on human cancer cells. Among the various organs, the toxin extracted from the liver showed potential antiproliferative activity at an inhibitory concentration of 50 µg/ml on HT-29 and MDA-MB231cell lines, 75 µg/ml on A549 and 100 µg/ml on AGS. The cellular and nuclear morphological changes, induction of apoptosis, and its stages were observed microscopically through different staining methods. Further, the DNA damage was confirmed by DNA fragmentation analysis to confirm apoptosis. The results suggest that the extract of toxin (liver) shows a significant antiproliferative effect on the human cancer cell lines.