Hepatocytes Research Articles

In vitro antitumor effects of methanolic extracts of three Ganoderema mushrooms

Ganoderma mushrooms have a variety of pharmacological activities and may have antitumor effects. Therefore, the antitumor activity of the methanolic fruiting body extracts of three Ganoderma spp. will be evaluated by estimating cell viability, cell cycle parameters and the mode of cellular death. In this regard, Sulfo-rhodamine B staining and flow cytometry were used. Hepatocellular carcinoma (HepG2) and breast ductal carcinoma (T-47D) cell lines were used as cancer models, while mouse normal liver (BNL) and oral epithelial cell (OEC) lines were used as respective controls. The results revealed that Ganoderma resinaceum extract decreased the viability of BNL at an IC50 > 100 µg/mL but not that of HepG2 at an IC50 of 72.32 µg/mL. Additionally, Ganoderma australe and Ganoderma mbrekobenum decreased the viability of OEC cell line at an IC50 of 328.29 and 271.56 µg/ mL, respectively. On the other hand, the IC50 of T-47D were 221.95 and 236.45 µg/mL, respectively. The three extracts arrested the cell life cycle at the G1 phase in each case. G. resinaceum extract stimulated total apoptosis (Q2 + Q4) of 19.99% with low necrosis (Q1). However, the percentages of total cell necrosis in the T-47D cell line treated with the other two extracts were 31.10% and 18.28%, respectively while the percentages of total cell apoptosis were 6.83% and 1.78%, respectively. Thus, G. resinaceum significantly inhibited the viability of the HepG2 cell line, while both the G. australe and G. mbrekobenum extracts significantly decreased the viability of the T-47D cell line. These results may encourage speculation about their possible use for the therapeutic management of hepatocellular carcinoma and breast ductal carcinoma after further investigation.

A new Leydig cell-exclusive Cre line allows lineage tracing of both fetal and adult Leydig cell populations in the mouse.

Leydig cells produce hormones that are required for male development, fertility, and health. Two Leydig cell populations produce these hormones but at different times during development: fetal Leydig cells which are active during fetal life and adult Leydig cells that are functional postnatally. Historically, our ability to understand the origin and function of Leydig cells has been made difficult by the lack of genetic models to exclusively target these cells. Taking advantage of the Leydig cell-exclusive expression pattern of the Insl3 gene, we used a CRISPR/Cas9 gene editing strategy to knock-in iCre recombinase into the mouse Insl3 locus. To demonstrate the Leydig cell-exclusive nature of our iCre line, lineage tracing experiments were performed by crossing Insl3iCre mice with a Rosa26LoxSTOPLox-TdTomato reporter. iCre activity was restricted to male offspring. TdTomato fluorescence was detected in both fetal and adult Leydig cells and co-localized with CYP17A1, a classic Leydig cell marker. Prior to birth, fluorescence was observed in fetal Leydig cells beginning at embryonic day 13.0. Fluorescence was also detected in adult Leydig cells starting at postnatal day 5 and continuing to the mature testis. Fluorescence was not detected in any other fetal or adult tissue examined, except for the unexpected finding that the adrenal cortex contains some Insl3-expressing Leydig-like cells. Our Leydig cell-exclusive iCre line therefore constitutes an invaluable new tool to study not only the origin of Leydig cells but also to target genes that have been long-proposed to be important for the development and functioning of these critical endocrine cells.

Cell-free supernatants from plant growth-promoting rhizobacteria Bacillus albus strains control Aspergillus flavus disease in peanut and maize seedlings

BackgroundAspergillus flavus, a seed-borne fungal pathogen, colonizes host plants and exploits nutrients, hindering the growth of seedlings such as peanut and maize. This study investigates the effectiveness of cell-free supernatants (CFSs) from the plant growth-promoting rhizobacteria (PGPR) Bacillus albus strains NNK24 and NDP61, which belong to the Bacillus cereus group, in suppressing A. flavus AF1.ResultsThe antifungal activity of these CFSs was attributed to their surfactant properties and chemical composition. These were characterized using rapid chemical assays and ultra-high-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight/mass spectrometry (UHPLC-ESI-QTOF/MS), combined with bioinformatic tools such as Global Natural Product Social Molecular Networking (GNPS) and Natural Products Atlas (NPAtlas). Identified putative antifungal compounds included two diketopiperazines (cyclo(Pro-Leu) and cyclo(2-hydroxy-Pro-Leu)), four macrolactins (7-O-succinyl macrolactin A, 7-O-methyl-5′-hydroxy-3′-heptenoate-macrolactin, macrolactin B, and macrolactin C), two siderophores (petrobactin and bacillibactin), and three cyclic lipopeptides (kurstakin 1, 2 or 3, and 4). These compounds are hypothesized to act synergistically via multiple mechanisms, including disruption of fungal membranes, iron capture, direct antibiosis, and triggering plant immunity. Both CFSs strongly suppressed the harmful effects of A. flavus AF1 and seed-borne A. flavus on peanut and maize seedlings, reducing disease incidence (DI) and disease severity index (DSI) compared to controls. The disease control efficacy (DCE) of the CFSs was comparable to that of the commercial fungicide. Additionally, the CFSs enhanced seed germination, vigor, seedling length, and weight in both peanut and maize. Vigor index (VI) values increased by 222.4–286.0% in peanuts and 181.7–216.4% in maize at 7 days after treatment (DAT).ConclusionCFSs of B. albus NNK24 and NDP61 show significant potential as bioprotective agents for sustainable agriculture. Importantly, their use eliminates the need for live bacterial cells from the B. cereus group, addressing biosafety concerns.Graphical abstract

Exploring the Unique Properties and Superior Schwann Cell Guiding Abilities of Spider Egg Sac Silk.

Spider silk (SPSI) is a promising candidate for use as a filler material in nerve guidance conduits (NGCs), facilitating peripheral nerve regeneration by providing a scaffold for Schwann cells (SCs) and axonal growth. However, the specific properties of SPSI that contribute to its regenerative success remain unclear. In this study, the egg sac silk of Trichonephila (T.) inaurata is investigated, which contains two distinct fiber types: tubuliform (TU) and major ampullate (MA) silk. These fibers serve as models to derive material parameters governing SC migration on natural silk substrates, since they are produced by the same spider, yet exhibiting distinct composition and morphology. In this paper, detailed characterization of the fibers' material properties and in vitro evaluation of their SC-guiding performance were conducted. Live cell imaging revealed significantly enhanced SC mobility and directionality on TU silk compared to MA silk, which is remarkable, given the lack of studies on TU silk for nerve regeneration. Our results suggest that the distinct morphological and material properties of these fibers are critical to their nerve-guiding potential. These insights contribute to the optimization of NGC filler materials by identifying key parameters essential for effective nerve regeneration.

Protocol for the Generation and 3D Culture of Fluorescently Labeled Multicellular Spheroids.

Spheroid culture systems have been extensively used to model the three-dimensional (3D) behavior of cells in vitro. Traditionally, spheroids consist of a single cell type, limiting their ability to fully recapitulate the complex inter-cellular interactions observed in vivo. Here we describe a protocol for generating cocultured spheroids composed of two distinct cell types, embedded within a 3D extracellular matrix (ECM) to better study cellular interactions. Fluorescent labeling of each cell type enables clear distinction and visualization, facilitating the analysis of cell invasion, proliferation, and behavior within the matrix. This method is particularly suited for studying matrix invasion, an essential process in cancer metastasis, using both fixed and live cell microscopy. The protocol is versatile and can be adapted for various cell types, providing a robust platform for investigating cell-cell interactions in cancer research, tissue remodeling, and drug screening.

The Rickettsia actin-based motility effectors RickA and Sca2 contribute differently to cell-to-cell spread and pathogenicity.

Rickettsia parkeri is an obligate intracellular, tick-borne bacterial pathogen that can cause eschar-associated rickettsiosis in humans. R. parkeri invades host cells, escapes from vacuoles into the cytosol, and undergoes two independent modes of actin-based motility mediated by effectors RickA or Sca2. Actin-based motility of R. parkeri enables bacteria to enter protrusions of the host cell plasma membrane that are engulfed by neighboring host cells. However, whether and how RickA and Sca2 independently contribute to cell-to-cell spread in vitro or pathogenicity in vivo has been unclear. Using live cell imaging of rickA::Tn and sca2::Tn mutants, we discovered both RickA and Sca2 contribute to different modes of cell-to-cell spread. Compared with Sca2-spread, RickA-spread involves the formation of longer protrusions that exhibit larger fluctuations in length and take a longer time to be engulfed into neighboring cells. We further compared the roles of RickA and Sca2 in vivo following intradermal (i.d.) infection of Ifnar1-/-; Ifngr1-/- mice carrying knockout mutations in the genes encoding the receptors for IFN-I (Ifnar1) and IFN-γ (Ifngr1), which exhibit eschars and succumb to infection with wild-type (WT) R. parkeri. We observed that RickA is important for severe eschar formation, whereas Sca2 contributes to larger foci of infection in the skin and dissemination from the skin to the internal organs. Our results suggest that actin-based motility effectors RickA and Sca2 drive two distinct forms of cell-to-cell spread and contribute differently to pathogenicity in the mammalian host.IMPORTANCERickettsia parkeri, a bacterium in the spotted fever group of Rickettsia species, can be transmitted from ticks to humans, leading to symptoms including fever, rash, muscle aches, and a lesion at the site of the tick bite. During Rickettsia parkeri infection, bacteria invade cells within the animal host, proliferate in the host cell's cytosol, move using a process called actin-based motility, and spread to neighboring host cells. Rickettsia parkeri is unusual in having two bacterial proteins that mediate actin-based motility. The significance of our research is to reveal that each of these bacterial actin-based motility proteins contributes differently to spread between cells and to the signs of infection in a mouse model of spotted fever disease. Our results are important for understanding the contribution of actin-based motility to mammalian infection by Rickettsia parkeri as well as to infection by other bacterial and viral pathogens that require this process to spread between cells and cause disease.

Insights into the toxic impact of long-term exposure to diethyl phthalate on commercially important species Catla (Labeo catla)

BackgroundDiethyl phthalate (DEP), used as a plasticizer, is more prevalent in the aquatic environment due to its widespread usage in plastics, cosmetics, and numerous pharmaceutical industries. It is a potential endocrine disruptor, but the underlying mechanism in fish needs to be investigated. The present study evaluated the toxic impacts of DEP exposure for 30 days on commercially important fish Labeo catla (Catla). Alterations at multiple level endpoints of the hypothalamic–pituitary–gonadal axis (HPG axis) were assessed. DEP-induced changes in oxidative stress, histopathological changes (liver, kidney and brain) and bioaccumulation in fish muscle were also evaluated.ResultsDEP exposure to 1/10th (1.62 mg/L) and 1/50th (0.32 mg/L) LC50 dose for 30 days to Catla revealed that it stimulated the expression of kisspeptin (Kiss 1 and Kiss 2) genes in the hypothalamus, leading to an increased GnRH concentration (36.75%) in the higher dose. The brain FSH levels increased by 11.24 and 55.42% times than control in both the doses. This led to an increase in circulating sex steroids E2 (41.62%) and 11 KT (24.59%) and eventually triggered hepatic Vtg production (23.90%) in a dose-dependent manner. DEP exposure lowered the concentration of antioxidants superoxide dismutase (SOD) and catalase (CAT), the effect being more pronounced in the higher dose. The histopathological alterations, such as hepatocyte vacuolization, sinusoidal congestion, loss of brush border, degeneration of lumen, infiltration of eosinophilic cells in liver, kidney and brain, respectively, were noted.ConclusionsThis pioneering study could provide detailed insight into the endocrine disruptive potential of DEP in fish, as evidenced by its impact at multiple endpoints, even at low doses after 30 days of exposure. The output of this investigation thus emphasized the need for regular monitoring of DEP for ecological risk assessment.

Exploring the Mechanisms of Iron Overload-Induced Liver Injury in Rats Based on Transcriptomics and Proteomics

Iron is a trace element that is indispensable for the growth and development of animals. Excessive iron supplementation may lead to iron overload and elevated reactive oxygen species (ROS) production in animals, causing cellular damage. Nevertheless, the precise mechanism by which iron overload causes cell injury remains to be fully elucidated. In this study, 16 male SD rats aged 6 to 7 weeks were randomly assigned to either a control group (CON) or an iron overload group (IO). Rats in the iron overload group received 150 mg/kg iron dextran injections every three days for a duration of four weeks. The results indicated that iron treatment with iron dextran significantly increased the scores of steatosis (p < 0.05) and inflammation (p < 0.05) in the NAS score. The integrated transcriptomic and proteomic analysis suggests that HO-1 and Lnc286.2 are potentially significant in iron overload-induced liver injury in rats. In vitro experiments utilizing ferric ammonium citrate (FAC) were conducted to establish an iron overload model in rat liver-derived BRL-3A cells. The result found that FAC treatment can significantly increase the BRL-3A cell’s Fe2+ content (p < 0.05), ROS (p < 0.01), lipid ROS (p < 0.01) levels, and the expression of the HO-1 gene and protein (p < 0.01), aligning with proteomic and transcriptomic findings. HO-1 inhibition can significantly decrease BRL-3A cell vitality (p < 0.01) and promote ROS (p < 0.05) and lipid ROS (p < 0.01), thus aggravating FAC-induced BRL-3A cell iron overload damage. Using the agonist of HO-1 agonist cobalt protoporphyrin (CoPP) to induce HO-1 overexpression can significantly alleviate the decrease in FAC-induced BRL-3A cell viability (p < 0.01), ROS (p < 0.01), and lipid ROS (p < 0.01). In addition, siLnc286.2 treatment can increase HO-1 expression, alleviate the decline of FAC-induced BRL-3A cell activity, and increase lipid ROS (p < 0.05) content. In conclusion, the findings of this study suggest that by suppressing the expression of Lnc286.2, we can enhance the expression of HO-1, which in turn alleviates lipid peroxidation in cells and increases their antioxidant capacity, thereby exerting a protective effect against liver cell injury induced by iron overload.

Rhamnogalacturonan promotes intestinal mucosal repair through increased cell migration.

Mucosal healing is the primary goal for Inflammatory Bowel Diseases (IBD) treatment. We previously showed the direct beneficial effects of rhamnogalacturonan (RGal) on intestinal epithelial barrier function. Here, we aimed to evaluate the effect of RGal in intestinal epithelial wound healing. Confluent cancer cell lines and colonoid monolayers were wounded, treated with RGal for 48 h and assessed using a live cell imaging system. Proliferation and apoptosis of cells were evaluated using EdU and TUNEL assays, respectively. Antagonists and inhibitors were used to determine the receptor and signaling pathways involved. Female and male mice with DSS-induced colitis were treated orally with RGal for 7 days during the recovery phase. RGal enhanced wound healing in Caco-2, T84 and primary cells by increasing cell migration. Inhibition of pre-transcriptional signaling pathways FAK, Src, PI3K, Rho family, and JNK reversed the RGal-induced wound healing. RNAseq data from Caco-2 and primary cells treated with RGal showed the upregulation of NF-κB pathway at 12 h. Actinomycin D, Bay 11-7082 or JSH-23, and NS-398 treatment significantly reversed the effect of RGal on wound healing, confirming that the response was also transcriptionally dependent and involved NF-κB signaling and downstream COX-2 protein activity. RGal treatment of male mice enhanced recovery from DSS colitis. RGal promoted wound healing in cancer and primary cells by increasing cell migration and accelerated epithelial mucosal healing in male mice. Our findings show a novel mechanism of action of RGal in wound healing that could help in mucosal healing and the resolution of intestinal inflammation.

Synthesis and Characterization of N-Doped Carbon Quantum Dots and its Application for Efficient Delivery of Curcumin in Live Cell.

To improve bioavailability, enhance the solubility and stability of the hydrophobic drug curcumin, nanoparticles such as carbon quantum dots (CQDs) are unique choices. In this study, we present a simple, cost-effective, and eco-friendly method for synthesizing nitrogen-doped carbon quantum dots (N-CQDs) and their application in the efficient delivery of hydrophobic drugs curcumin into live cancer cells. The N-CQDs produced in this study exhibit excellent water solubility, remarkable stability, and high biocompatibility. To synthesize the N-CQD, we use a carbon source found naturally (lemon juice) and for doping, we use N-rich doping agents such as ethylene diamine and urea by using eco-friendly chemical oxidation methods. The resulting N-CQDs, with particle sizes under 10 nm, exhibit a good quantum yield, reinforcing their utility for biomedical applications. N-CQDs and drug-loaded particles are evaluated using various techniques like UV-Vis, Fluorescence Spectroscopy, Dynamic Light Scattering (DLS), and Atomic Force Microscopy (AFM) as well. Additionally, we report a remarkable method to use N-CQDs as carriers for the anticancer drug curcumin, significantly enhancing the solubility in live cells. Our research also delved into the application of N-CQDs in in vivo bioimaging and drug release studies within live cancer cells, with a particular focus on their pH-dependence behavior.

A gene-encoded bioprotein second harmonic generation (SHG) probe from Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin for live cell imaging.

Compared to fluorescence, second harmonic generation (SHG) has recently emerged as an excellent signal for imaging probes due to its unmatched advantages in terms of no photobleaching, no phototoxicity, no signal saturation, as well as the superior imaging accuracy with excellent avoidance of background noise. Existing SHG probes are constructed from heavy metals and are cellular exogenous, presenting with high cytotoxicity, difficult cellular uptake, and the limitation of non-heritability. We, therefore, initially propose an innovative gene-encoded bioprotein SHG probe derived from Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin. The primitive gene of AcMNPV polyhedrin was codon-optimized and mutated in its nuclear localization sequence to achieve cytoplasmic expression in mammalian cells. While providing strong SHG signals, this gene-modified AcMNPV (GM-AcMNPV) polyhedrin could be utilized as an SHG probe for cell imaging. Our experimental results demonstrated successful expression of GM-AcMNPV polyhedrin in the cytoplasm of HEK293T cells and bone mesenchymal stem cells (BMSCs), and verified its characteristic features as an SHG probe. Such SHG probes exhibit high biocompatibility and showed no hindering of central physiological activities such as the differentiation of stem cells. Most importantly, our SHG probes may be successfully used for imaging in living cells. This work will inspire the development of gene encoding-derived bioprotein SHG probes, for long-term tracing of cells/stem cells along with their division, to understand stem cell cycles, reveal stem cell-based therapy mechanisms in regenerative medicine, and unravel cell lineage origins and fates in developmental biology, among other potential applications.

Super-photostable organic dye for long-term live-cell single-protein imaging.

Organic dyes play a crucial role in live-cell imaging because of their advantageous properties, such as photostability and high brightness. Here we introduce a super-photostable and bright organic dye, Phoenix Fluor 555 (PF555), which exhibits an order-of-magnitude longer photobleaching lifetime than conventional organic dyes without the requirement of any anti-photobleaching additives. PF555 is an asymmetric cyanine structure in which, on one side, the indole in the conventional Cyanine-3 is substituted with 3-oxo-quinoline. PF555 provides a powerful tool for long-term live-cell single-molecule imaging, as demonstrated by the imaging of the dynamic single-molecule interactions of the epidermal growth factor receptor with clathrin-coated structures on the plasma membrane of a live cell under physiological conditions.

Epigenetic effects of four nutraceutical products on human liver cells

Background: There are numerous botanical extracts and fractions that have been shown to have a diverse range of bioactivities. Many nutraceutical products and food supplements contain phytochemicals - often multiple numbers of them as constituents of a single product. There is a paucity of data on the net bioactivities of these multi-component products. This paper reports the effects of four such products on the expression of six genes that are critical to the maintenance of good health. Objectives: We investigated several products that contain a number of constituents derived from plant species as well as essential minerals on the biochemical activities of liver cells in culture. The combination of these bioactives into one product results in multiple strong biological effects. Examples of these are presented in this paper with a discussion of how the constituents have provided this effect. Methods: The expression of six genes (ppar-α, glp-1, bdnf, sirt-1, nrf1, sod-1) in a human liver cell line (THLE-2 cells) were cultured for 24 hours in the presence of Vitalité, Reviv, Revíve and Collagène (products from THREE International). The RNA was isolated from these cells and from the control unsupplemented cells. The levels of transcripts of these genes in these isolates were measured by RT-PCR. The changes in the concentrations of the mRNA from the supplemented cells were compared with those from reference cells. Results: Of the four products, Revíve boosted the transcription of the glp-1, the bdnf, the sirt-1, the nrf1, and the sod-1 genes. Éternel boosted the transcription of ppar-α, and the sod-1 genes. Vitalité increased the transcription of the glp-1, the bdnf, the nrf1, and the sod-1 genes. Collagène stimulated the expression of the bdnf, and the sod-1 genes. The use of nutraceutical products, either as a complement or as a substitute for pharmaceuticals, has been receiving increased attention for maintaining good health. These products are multi-component. Evidence for their clinical efficacy is frequently lacking. Conclusion: The results of this project provide evidence that the phytochemicals in the four products investigated can provide the bio-effects that regulate the expression of key genes that control fundamental essential metabolic activities and so can justify the claims made for them. Keywords: Epigenetics, nutritional supplements, phytonutrients, liver health

Fe3O4-viral-like Mesoporous Silica Nanoparticle(Fe3O4-vMSN)-Sustained Release of Lenvatinib for Targeted Treatment of Hepatocellular Carcinoma

Background: Lenvatinib is an oral tyrosine kinase inhibitor that selectively inhib-its receptors involved in tumor angiogenesis and tumor growth. It is an emerging first-line treatment agent for hepatocellular carcinoma (HCC). However, there is no intravenous ad-ministration of Lenvatinib. Aims: This study aimed to construct nanocomposites that can efficiently support Lenvatinib and target liver cancer tissues and cells. Objective: In this study, ferric oxide-viral-like mesoporous silica nanoparticles-folic acid (Fe3O4-vMSN-FA) nanocomposites loaded with Lenvatinib were constructed, and their anti-hepatocellular carcinoma effects were evaluated. Methods: The hydrothermal method was used to synthesize ferric oxide (Fe3O4). Ferric ox-ide-viral-like mesoporous silica nanoparticles (Fe3O4-vMSN) were synthesized using a two-phase method. Then, Fe3O4-vMSN was modified with folic acid (Fe3O4-vMSN-FA) to better target tumor cells. Results: The experimental data showed that Fe3O4-vMSN-FA nanocomposites were suc-cessfully synthesized and could be loaded with Lenvatinib (Len@ Fe3O4-vMSN-FA). Fe3O4-vMSN-FA had good stability and biocompatibility, and it can release the loaded Len-vatinib faster in an acidic environment (pH 5.5). CCK8 assay and flow cytometry showed that HepG2 cells in the Len@ Fe3O4-vMSN group had the lowest cell viability and the high-est apoptosis rate, confirming the anticancer properties of Len@ Fe3O4-vMSN-FA in vitro. In addition, transwell experiments showed that the migration and invasion ability of HepG2 cells in the Len@ Fe3O4-vMSN-FA group were significantly inhibited. In vivo fluorescence imaging in mice confirmed the enhanced tumor-targeting ability of Fe3O4-vMSN-FA. The tumor volume of the Len@ Fe3O4-vMSN-FA group was significantly reduced, and there was no significant effect on body weight. Moreover, serum liver function index (ALT and AST) and HE staining showed that Len@ Fe3O4-vMSN-FA did not cause obvious damage to organ tissue. Conclusion: Len@ Fe3O4-vMSN-FA has a good anti-liver cancer effect. Fe3O4-vMSN-FA can be used as an alternative platform for MSCs for drug delivery, providing more options for cancer therapy.

Lipocalin 2 in Obesity and Diabetes: Insights into Its Role in Energy Metabolism

Background: Lipocalin 2 (LCN2), also known as neutrophil gelatinase-associated lipocalin, is a 25 kDa protein involved in immune defense, inflammation, and metabolism. Results: LCN2 is widely expressed across various tissues, including immune cells, bone, adipose tissue, liver, kidneys, lung, spleen, and epithelial cells, and exhibits sex- and fat depot-specific expression patterns. Structurally, LCN2 contains a hydrophobic lipid-binding pocket and glycosylation sites, enabling it to interact with diverse ligands and form dimers. In innate immunity, LCN2 plays a critical role by sequestering iron-laden siderophores, thereby restricting bacterial growth. Beyond its role in infection control, LCN2 is implicated in metabolic inflammation and diseases such as obesity and diabetes. Recent research has highlighted a pivotal role for LCN2 in mitochondrial phospholipid metabolism and mitochondrial function. In metabolic diseases and mitochondrial metabolism, LCN2 appears to display paradoxical effects. While some studies link it to improved insulin sensitivity, glucose regulation, and mitochondrial function, others associate it with insulin resistance, obesity, and mitochondrial dysfunction. These inconsistencies may arise from differences in experimental conditions and study populations. Conclusions: This review provides an up-to-date summary of LCN2’s multifaceted roles in obesity, diabetes, energy balance, and mitochondrial function, emphasizing its context-dependent effects. LCN2 appears to have dual roles, exerting both protective and detrimental outcomes depending on the physiological or pathological context, sex, cell types, and experimental conditions. Further research is necessary to unravel its complex functions and resolve conflicting findings, particularly in metabolic disorders.

Modulatory effect of quercetin on aspirin-induced hepatoxicity in Wistar rats

Objectives: Aspirin, also known as acetylsalicylic acid, is a non-steroidal anti-inflammatory drug medication. Aspirin has been shown to have a wide range of pharmacological effects, such as antipyretic, antiplatelet and analgesic effects. Although there is little known about how aspirin causes hepatotoxicity at the cellular level, this does happen and there is a need to look into some hepatoprotective remedies. The aim of this study was to assess how quercetin (QE) affects aspirin-induced hepatotoxicity in Wistar rats. Materials and Methods: Thirty-five male adult Wistar rats divided into seven experimental groups were used in this study. These groups received different treatments: Some were given varying concentrations of QE (30 mg/kg and 60 mg/kg), while others received aspirin (50 mg/kg). In addition, there were control groups that did not receive either aspirin or QE, with normal saline and corn oil being administered instead. The administration of treatments lasted for 30 days, after which the experiment was concluded, and the rats’ livers were removed for histological examination. Simultaneously, blood serum samples were collected for the biochemical analysis of liver enzyme markers. The level of significance was set at 0.05, and the data collected was analysed using the Statistical Package for the Social Sciences version 29. Results: It was found that aspirin increased the level of liver enzyme markers in the serum after 30 days of administration resulting from damage to the liver cells, this effect was most significant at an aspirin concentration of 50 mg/kg and QE at a concentration of 60 mg/kg was most effective in exhibiting hepatoprotective potentials. Conclusion: The results suggest that aspirin 50 mg/kg could be hepatotoxic due to the lessening of antioxidant effects, and QE has a modulating effect on aspirin-induced toxicity.

The small GTPase MRAS is a broken switch

Intense research on founding members of the RAS superfamily has defined our understanding of these critical signalling proteins, leading to the premise that small GTPases function as molecular switches dependent on differential nucleotide loading. The closest homologs of H/K/NRAS are the three-member RRAS family, and interest in the MRAS GTPase as a regulator of MAPK activity has recently intensified. We show here that MRAS does not function as a classical switch and is unable to exchange GDP-to-GTP in solution or when tethered to a lipid bilayer. The exchange defect is unaffected by inclusion of the GEF SOS1 and is conserved in a distal ortholog from nematodes. Synthetic activating mutations widely used to study the function of MRAS in a presumed GTP-loaded state do not increase exchange, but instead drive effector binding due to sampling of an activated conformation in the GDP-loaded state. This includes nucleation of the SHOC2-PP1Cα holophosphatase complex. Acquisition of NMR spectra from isotopically labeled MRAS in live cells validated the GTPase remains fully GDP-loaded, even a supposed activated mutant. These data show that RAS GTPases, including those most similar to KRAS, have disparate biochemical activities and challenge current dogma on MRAS, suggesting previous data may need reinterpretation.

Developing Orthogonal Fluorescent RNAs for Photoactive Dual-color Imaging of RNAs in Live Cells.

Fluorogenic RNA aptamers have revolutionized the visualization of RNAs within complex cellular processes. A representative category of them employs the derivatives of green fluorescent protein chromophore, 4-hydroxybenzlidene imidazolinone (HBI), as chromophores. However, the structural homogeneity of their chromophoric backbones causes severe cross-reactivity with other homologous chromophores. This limitation impairs their multiplexing capabilities, which are essential for the simultaneous visualization of multiple RNA species in live cells. Herein, we rationally designed a series of red-shifted chromophores and employed SELEX-independent engineering to develop a novel fluorogenic RNA aptamer, mSquash. mSquash displays specific and intense fluorescence upon binding with our red-shifted chromophore DFHBFPD (Ex/Em = 501/624 nm). The mSquash/DFHBFPD allows orthogonal imaging of selected RNA targets alongside the established Broccoli/DFHBI-1T (Ex/Em = 472/501 nm), facilitating multiplexed live cell imaging of various targets. Moreover, we expanded the application of fluorescent RNA to photoactive imaging by constructing two genetically encoded photoactivatable fluorescent RNAs for the first time. This innovative approach allows photoactivatable control of fluorescent RNAs via specific light wavelengths (365 nm and 450 nm), enabling spatiotemporal dual-color imaging of RNAs in live cells. Our findings represent a significant advancement in fluorescent RNA-based orthogonal imaging and spatiotemporal analysis of RNAs.

Developing Orthogonal Fluorescent RNAs for Photoactive Dual‐color Imaging of RNAs in Live Cells

Fluorogenic RNA aptamers have revolutionized the visualization of RNAs within complex cellular processes. A representative category of them employs the derivatives of green fluorescent protein chromophore, 4‐hydroxybenzlidene imidazolinone (HBI), as chromophores. However, the structural homogeneity of their chromophoric backbones causes severe cross‐reactivity with other homologous chromophores. This limitation impairs their multiplexing capabilities, which are essential for the simultaneous visualization of multiple RNA species in live cells. Herein, we rationally designed a series of red‐shifted chromophores and employed SELEX‐independent engineering to develop a novel fluorogenic RNA aptamer, mSquash. mSquash displays specific and intense fluorescence upon binding with our red‐shifted chromophore DFHBFPD (Ex/Em = 501/624 nm). The mSquash/DFHBFPD allows orthogonal imaging of selected RNA targets alongside the established Broccoli/DFHBI‐1T (Ex/Em = 472/501 nm), facilitating multiplexed live cell imaging of various targets. Moreover, we expanded the application of fluorescent RNA to photoactive imaging by constructing two genetically encoded photoactivatable fluorescent RNAs for the first time. This innovative approach allows photoactivatable control of fluorescent RNAs via specific light wavelengths (365 nm and 450 nm), enabling spatiotemporal dual‐color imaging of RNAs in live cells. Our findings represent a significant advancement in fluorescent RNA‐based orthogonal imaging and spatiotemporal analysis of RNAs.

Applications and Challenges of RNA Interference Technology in Therapeutic Development

RNA interference (RNAi) is a potent biological mechanism enabling the targeted silencing of specific genes, transforming gene regulation and therapeutic approaches in medical science. Using small RNA molecules such as small interfering RNAs (siRNAs) and microRNAs (miRNAs), RNAi allows for precise mRNA degradation or translational repression. Clinically, RNAi has been applied to treat genetic disorders such as hereditary transthyretin amyloidosis (hATTR) and acute hepatic porphyria (AHP). RNAi also holds promise in cancer therapies, targeting oncogenes like KRAS and pathways such as PI3K/AKT/mTOR to slow tumor growth. However, several challenges limit RNAi's broader clinical adoption. A significant barrier is the development of effective delivery systems beyond liver cells, as current methods, such as lipid nanoparticles, have limitations in targeting other tissues. Furthermore, issues such as immune activation, off-target effects, and ensuring RNAi stability must be addressed. Regulatory hurdles concerning long-term safety and gene-silencing specificity also pose challenges. Looking ahead, advancements in next-generation RNAi molecules and delivery platforms, alongside combination therapies, offer promising solutions to overcome these limitations. his review explores the fundamental mechanisms of RNAi, including its role in gene regulation and gene silencing, and highlights its clinical applications in treating genetic disorders, cancers, and viral infections