Search Results (21)

Mitochondrial transplantation (MTx) has emerged as a potential therapeutic approach for diseases associated with mitochondrial dysfunction, yet its scalability and cross-species feasibility remain underexplored. This study aimed to evaluate the dose-dependent uptake and molecular effects of xenogeneic mitochondrial transplantation (xeno-MTx) using rat-derived mitochondria in mouse neuronal systems. HT-22 hippocampal neuronal cells and a murine model of cardiac arrest-induced global cerebral ischemia were used to assess mitochondrial uptake, gene expression, and mitochondrial DNA presence. Donor mitochondria were isolated from rat pectoralis muscle and labeled with MitoTracker dyes. Flow cytometry and confocal microscopy revealed a dose-dependent increase in donor mitochondrial uptake in vitro. Quantitative PCR demonstrated a corresponding increase in rat-specific mitochondrial DNA and upregulation of Mfn2 and Bak1, with no changes in other fusion, fission, or apoptotic genes. Inhibitor studies indicated that mitochondrial internalization may involve actin-dependent macropinocytosis and cholesterol-sensitive endocytic pathways. In vivo, rat mitochondrial DNA was detected in mouse brains post–xeno-MTx, confirming donor mitochondrial delivery to ischemic tissue. These findings support the feasibility of xeno-MTx and its dose-responsive biological effects in neuronal systems while underscoring the need for further research to determine long-term functional outcomes and clinical applicability. Full article

Background: Tay–Sachs disease (TSD) is an autosomal recessive lysosomal storage disorder characterized by the accumulation of GM2 ganglioside due to mutations in the HEXA gene, which encodes the α-subunit of β-Hexosaminidase A. This accumulation leads to significant neuropathological effects and premature death in affected individuals. No effective treatments exist, but enzyme replacement therapies are under investigation. In our previous work, we demonstrated the internalization and efficacy of human recombinant lysosomal β-hexosaminidase A (rhHex-A), produced in the methylotrophic yeast Pichia pastoris, in reducing lipids and lysosomal mass levels in fibroblasts and neural stem cells derived from patient-induced pluripotent stem cells (iPSCs). In this study, we further evaluated the potential of rhHex-A to prevent GM2 accumulation using fibroblast and neuroglia cells from a TSD patient alongside a relevant mouse model. Methods: Fibroblasts and neuroglial cell lines derived from a murine model and TSD patients were treated with 100 nM rhHexA for 72 h. After treatment, cells were stained by anti-GM2 (targeting GM2 ganglioside; KM966) and anti-LAMP1 (lysosomal-associated membrane protein 1) colocalization staining and incubated with 50 nM LysoTracker Red DND-99 to label lysosomes. In addition, GM2AP and HEXB expression were analyzed to assess whether rhHex-A treatment affected the levels of enzymes involved in GM2 ganglioside degradation. Results: Immunofluorescence staining for LysoTracker and colocalization studies of GM2 and Lamp1 indicated reduced lysosomal mass and GM2 levels. Notably, rhHex-A treatment also affected the expression of the HEXB gene, which is involved in GM2 ganglioside metabolism, highlighting a potential regulatory interaction within the metabolic pathway. Conclusions: Here, we report that rhHex-A produced in yeast can efficiently degrade GM2 ganglioside and rescue lysosomal accumulation in TSD cells. Full article

Ovarian aging significantly impacts female fertility, with mitochondrial dysfunction emerging as a key factor. This study investigated the effects of recombinant follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on mitochondrial function and metabolism in aging female reproductive cells. Human granulosa cells (HGL5) were treated with FSH/LH or not. Mitochondrial function was assessed through various assays, including mitochondrial mass, membrane potential, ROS levels, and ATP production. Mitochondrial dynamics and morphology were analyzed using MitoTracker staining. Cellular respiration was measured using a Seahorse Bioenergetics Analyzer. Metabolic reprogramming was evaluated through gene expression analysis and metabolite profiling. In vivo effects were studied using aging mouse oocytes. FSH/LH treatment significantly improved mitochondrial function in aging granulosa cells, increasing mitochondrial mass and membrane potential while reducing ROS levels. Mitochondrial dynamics showed a shift towards fusion and elongation. Cellular respiration, ATP production, and spare respiratory capacity were enhanced. FSH/LH-induced favorable alterations in cellular metabolism, favoring oxidative phosphorylation. In aging mouse oocytes, FSH/LH treatment improved in vitro maturation and mitochondrial health. In conclusion, FSH/LH supplementation ameliorates age-related mitochondrial dysfunction and improves cellular metabolism in aging female reproductive cells. Full article

Background: Liver cancer treatment encounters considerable therapeutic challenges, especially because hypoxic microenvironments markedly reduce sensitivity to chemotherapeutic agents. TFAM (mitochondrial transcription factor A) plays a crucial role in maintaining mitochondrial function. Oroxylin A (OA), a flavonoid with potential therapeutic properties, demonstrated prospects in cancer treatment. However, the mechanism of the sensitizing effect of OA on cancer cells has not been elucidated. Methods: MTT assays were utilized to evaluate a hypoxia-induced resistance model. Plate colony formation assays, TEM, and JC-1 staining were used to examine the effects of siTFAM on proliferation and mitochondrial damage of HepG2 cells. Cox8-EGFP-mCherry plasmid transfection, LysoTracker and MitoTracker colocalization analysis, and WB were conducted to evaluate the influence of OA on mitophagy. The effect of OA on p53 ubiquitination levels was investigated by Co-IP and the CHX chase assay. A mouse xenograft tumor model was utilized to assess the therapeutic effect of OA on HepG2 cells in vivo. Results: OA significantly improved the inhibitory effect of sorafenib by inhibiting mitophagy on HepG2 cells in in vitro and in vivo models. Notably, the molecular docking and thermal shift assays indicated a clear binding of OA and TFAM. Further research revealed that OA suppressed p53 acetylation and promoted its degradation by downregulating TFAM expression, which ultimately inhibited mitophagy in hypoxia. Conclusions: OA has demonstrated the potential to enhance the efficacy of sorafenib treatment for liver cancer, and TFAM may be one of its targets. Full article

Some of the barriers preventing virtual reality (VR) from being widely adopted are the cost and unfamiliarity of VR systems. Here, we propose that in many cases, the specialized controllers shipped with most VR head-mounted displays can be replaced by a regular smartphone, cutting the cost of the system, and allowing users to interact in VR using a device they are already familiar with. To achieve this, we developed SmartVR Pointer, an approach that uses smartphones to replace the specialized controllers for two essential operations in VR: selection and navigation by teleporting. In SmartVR Pointer, a camera mounted on the head-mounted display (HMD) is tilted downwards so that it points to where the user will naturally be holding their phone in front of them. SmartVR Pointer supports three selection modalities: tracker based, gaze based, and combined/hybrid. In the tracker-based SmartVR Pointer selection, we use image-based tracking to track a QR code displayed on the phone screen and then map the phone’s position to a pointer shown within the field of view of the camera in the virtual environment. In the gaze-based selection modality, the user controls the pointer using their gaze and taps on the phone for selection. The combined technique is a hybrid between gaze-based interaction in VR and tracker-based Augmented Reality. It allows the user to control a VR pointer that looks and behaves like a mouse pointer by moving their smartphone to select objects within the virtual environment, and to interact with the selected objects using the smartphone’s touch screen. The touchscreen is used for selection and dragging. The SmartVR Pointer is simple and requires no calibration and no complex hardware assembly or disassembly. We demonstrate successful interactive applications of SmartVR Pointer in a VR environment with a demo where the user navigates in the virtual environment using teleportation points on the floor and then solves a Tetris-style key-and-lock challenge. Full article

High levels of alcohol intake alter brain gene expression and can produce long-lasting effects. FK506-binding protein 51 (FKBP51) encoded by Fkbp5 is a physical and cellular stress response gene and has been associated with alcohol consumption and withdrawal severity. Fkbp5 has been previously linked to neurite outgrowth and hippocampal morphology, sex differences in stress response, and epigenetic modification. Presently, primary cultured Fkbp5 KO and WT mouse neurons were examined for neurite outgrowth and mitochondrial signal with and without alcohol. We found neurite specification differences between KO and WT; particularly, mesh-like morphology was observed after alcohol treatment and confirmed higher MitoTracker signal in cultured neurons of Fkbp5 KO compared to WT at both naive and alcohol-treated conditions. Brain regions that express FKBP51 protein were identified, and hippocampus was confirmed to possess a high level of expression. RNA-seq profiling was performed using the hippocampus of naïve or alcohol-injected (2 mg EtOH/Kg) male and female Fkbp5 KO and WT mice. Differentially expressed genes (DEGs) were identified between Fkbp5 KO and WT at baseline and following alcohol treatment, with female comparisons possessing a higher number of DEGs than male comparisons. Pathway analysis suggested that genes affecting calcium signaling, lipid metabolism, and axon guidance were differentially expressed at naïve condition between KO and WT. Alcohol treatment significantly affected pathways and enzymes involved in biosynthesis (Keto, serine, and glycine) and signaling (dopamine and insulin receptor), and neuroprotective role. Functions related to cell morphology, cell-to-cell signaling, lipid metabolism, injury response, and post-translational modification were significantly altered due to alcohol. In summary, Fkbp5 plays a critical role in the response to acute alcohol treatment by altering metabolism and signaling-related genes. Full article

Glaucoma is one of the leading causes of blindness worldwide. Decreased aqueous humor drainage causes an increase in intraocular pressure (IOP), which in turn damages the ganglion cells of the retina and optic nerve. A mouse model of glaucoma was used to examine the behavior of Myo/Nog (M/N) cells, which were previously shown to respond to cataract surgery and retinopathy induced by hypoxia, light damage, and intravitreal injection of human retinal pigment epithelial cells. M/N cells express the skeletal-muscle-specific transcription factor MyoD, the bone morphogenetic protein inhibitor Noggin, and brain-specific angiogenesis inhibitor 1 (BAI1). Glaucoma was induced by injecting microbeads into the anterior chamber (AC) of the right eye to obstruct the flow of aqueous humor into the trabecular meshwork. IOP was elevated within three days of addition of microbeads. Loss of retinal ganglion cells (RGCs) and thinning of the ganglion cell layer–nerve fiber layer (GCL-NFL) was observed in tissue sections by day 32. The injection of microbeads resulted in an increase in BAI1-positive (+) M/N cells in the trabecular meshwork, ciliary body, canal of Schlemm, cornea, and ganglion cell layer (GCL). M/N cells ingested microbeads. The effect of further increasing the population of M/N cells on IOP and RGC loss was determined by injecting BAI1+ cells isolated from the brain into the AC of both eyes. Exogenous M/N cells prelabeled with CellTracker™ Red were found in the same tissues as the endogenous population of M/N cells in eyes with and without elevated IOP. The addition of M/N cells did not significantly reduce IOP in bead-injected eyes. However, there were significantly more RGCs and the NFL was thicker in glaucomatous eyes with M/N cell supplementation than eyes injected with phosphate-buffered saline. The numbers of RGCs and NFL thickness were similar in glaucomatous and non-glaucomatous eyes after adding M/N cells. These results demonstrate that endogenous M/N cells respond to elevated IOP in the anterior and posterior segments in response to induction of glaucoma. M/N cells’ mitigation of RGC loss may reflect a neuroprotective effect within the retina, as opposed to a significant drop in IOP. Full article

Raster maps provide intuitive visualizations of remote sensing data representing various phenomena on the Earth’s surface. Reading raster maps with intricate information requires a high cognitive workload, especially when it is necessary to identify and compare values between multiple layers. In traditional methods, users need to repeatedly move their mouse and switch their visual focus between the map content and legend to interpret various grid value meanings. Such methods are ineffective and may lead to the loss of visual context for users. In this research, we aim to explore the potential benefits and drawbacks of gaze-adaptive interactions when interpreting raster maps. We focus on the usability of the use of low-cost eye trackers on gaze-based interactions. We designed two gaze-adaptive methods, gaze fixed and gaze dynamic adaptations, for identifying and comparing raster values between multilayers. In both methods, the grid content of different layers is adaptively adjusted depending on the user’s visual focus. We then conducted a user experiment by comparing such adaptation methods with a mouse dynamic adaptation method and a traditional method. Thirty-one participants (n = 31) were asked to complete a series of single-layer identification and multilayer comparison tasks. The results indicated that although gaze interaction with adaptive legends confused participants in single-layer identification, it improved multilayer comparison efficiency and effectiveness. The gaze-adaptive approach was well received by the participants overall, but was also perceived to be distracting and insensitive. By analyzing the participants’ eye movement data, we found that different methods exhibited significant differences in visual behaviors. The results are helpful for gaze-driven adaptation research in (geo)visualization in the future. Full article

Theories of embodied cognition suggest that hand motions and cognition are closely interconnected. An emerging technique of tracking how participants move a computer mouse (i.e., the mouse-tracking technique) has shown advantages over the traditional response time measurement to detect implicit cognitive conflicts. Previous research suggests that attention is essential for subliminal processing to take place at a semantic level. However, this assumption is challenged by evidence showing the presence of subliminal semantic processing in the near-absence of attention. The inconsistency of evidence could stem from the insufficient sensitivity in the response time measurement. Therefore, we examined the role of attention in subliminal semantic processing by analyzing participants’ hand motions using the mouse-tracking technique. The results suggest that subliminal semantic processing is not only enhanced by attention but also occurs when attention is disrupted, challenging the necessity of facilitated top-down attention for subliminal semantic processing, as claimed by a number of studies. In addition, by manipulating the color of attentional cues, our experiment shows that the cue color per se could influence participants’ response patterns. Overall, the current study suggests that attentional status and subliminal semantic processing can be reliably revealed by temporal–spatial features extracted from cursor motion trajectories. Full article

This article aims to present the authors’ perspective regarding the challenges and opportunities of mouse-tracking methodology while performing experimental research, particularly related to the map-reading process. We briefly describe existing metrics, visualization techniques and software tools utilized for the qualitative and quantitative analysis of experimental mouse-movement data towards the examination of both perceptual and cognitive issues. Moreover, we concisely report indicative examples of mouse-tracking studies in the field of cartography. The article concludes with summarizing mouse-tracking strengths/potential and limitations, compared to eye tracking. In a nutshell, mouse tracking is a straightforward method, particularly suitable for tracking real-life behaviors in interactive maps, providing the valuable opportunity for remote experimentation; even though it is not suitable for tracking the actual free-viewing behavior, it can be concurrently utilized with other state-of-the-art experimental methods. Full article

The COVID-19 pandemic has made the world focus on providing effective and fair online learning systems. As a consequence, this paper proposed a new intelligent, fair assessment of online examinations for virtual and remotely controlled laboratory experiments running through Laboratory Learning Systems. The main idea is to provide students with an environment similar to being physically present in a Laboratory while conducting practical experiments and exams and detecting cheating with high accuracy at a minimal cost. Therefore, an intelligent assessment module is designed to detect cheating students by analyzing their mouse dynamics using Artificial Intelligence. The mouse interaction behavior method was chosen because it does not require any additional resources, such as a camera and eye tribe tracker, to detect cheating. Various AI algorithms, such as KNN, SVC, Random Forest, Logistic Regression, XGBoost, and LightGBM have been used to classify student mouse behavior to detect cheating, and many metrics are used to evaluate their performance. Moreover, experiments have been conducted on students answering online laboratory experimentations while cheating and when answering the exams honestly. Experimental results indicate that the LightGBM AI algorithm achieves the best cheat detection results up to an accuracy of 90%, precision of 88%, and degree of separation of 95%. Full article

Supporting cells of oocytes, i.e., cumulus cells, control oocyte quality, which determines fertilization success. Therefore, the transformation of mature and immature cumulus cells (MCCs and ICCs, respectively) into dysmature cumulus cells (DCCs) with dead characteristics deteriorates oocyte quality. However, the molecular basis for this transformation remains unclear. Here, we explored the link between autophagic decline and cumulus transformation using cumulus cells from patients with infertility, female mice, and human granulosa cell-derived KGN cell lines. When human cumulus cells were labeled with LysoTracker probes, fluorescence corresponding to lysosomes was enhanced in DCCs compared to that in MCCs and ICCs. Similarly, treatment with the autophagy inhibitor chloroquine elevated LysoTracker fluorescence in both mouse cumulus cells and KGN cells, subsequently suppressing ovulation in female mice. Electron microscopy analysis revealed the proliferation of abnormal lysosomes in chloroquine-treated KGN cells. Conversely, the addition of an autophagy inducer, trehalose, suppressed chloroquine-driven problematic lysosomal anomalies and ameliorated ovulation problems. Our results suggest that autophagy maintains the healthy state of the supporting cells of human oocytes by suppressing the formation of lysosomes. Thus, our results provide insights into the therapeutic effects of trehalose on female fertility. Full article

Extracellular vesicles (EVs), released from all cells, are essential to cellular communication and contain biomolecular cargo that can affect recipient cell function. Studies on the effects of contractile activity (exercise) on EVs usually rely on plasma/serum-based assessments, which contain EVs from many different cells. To specifically characterize skeletal muscle–derived vesicles and the effect of acute contractile activity, we used an in vitro model where C2C12 mouse myoblasts were differentiated to form myotubes. EVs were isolated from conditioned media from muscle cells at pre-differentiation (myoblasts) and post-differentiation (myotubes) and also from acutely stimulated myotubes (1 h @ 14 V, C-Pace EM, IonOptix, Westwood, MA, USA) using total exosome isolation reagent (TEI, ThermoFisher (Waltham, MA, USA), referred to as extracellular particles [EPs]) and differential ultracentrifugation (dUC; EVs). Myotube-EPs (~98 nm) were 41% smaller than myoblast-EPs (~167 nm, p < 0.001, n = 8–10). Two-way ANOVA showed a significant main effect for the size distribution of myotube vs. myoblast-EPs (p < 0.01, n = 10–13). In comparison, myoblast-EPs displayed a bimodal size distribution profile with peaks at <200 nm and 400–600, whereas myotube-Eps were largely 50–300 nm in size. Total protein yield from myotube-EPs was nearly 15-fold higher than from the myoblast-EPs, (p < 0.001 n = 6–9). Similar biophysical characteristics were observed when EVs were isolated using dUC: myotube-EVs (~195 nm) remained 41% smaller in average size than myoblast-EVs (~330 nm, p = 0.07, n = 4–6) and had comparable size distribution profiles to EPs isolated via TEI. Myotube-EVs also had 4.7-fold higher protein yield vs. myoblast EVs (p < 0.05, n = 4–6). Myotube-EPs exhibited significantly decreased expression of exosomal marker proteins TSG101, CD63, ALIX and CD81 compared with myoblast-EPs (p < 0.05, n = 7–12). Conversely, microvesicle marker ARF6 and lipoprotein marker APO-A1 were only found in the myotube-EPs (p < 0.05, n = 4–12). There was no effect of acute stimulation on myotube-EP biophysical characteristics (n = 7) or on the expression of TSG101, ARF6 or CD81 (n = 5–6). Myoblasts treated with control or acute stimulation–derived EPs (13 µg/well) for 48 h and 72 h showed no changes in mitochondrial mass (MitoTracker Red, ThermoFisher, Waltham, MA, USA), cell viability or cell count (n = 3–4). Myoblasts treated with EP-depleted media (72 h) exhibited ~90% lower cell counts (p < 0.01, n = 3). Our data show that EVs differed in size, distribution, protein yield and expression of subtype markers pre vs. post skeletal muscle–differentiation into myotubes. There was no effect of acute stimulation on biophysical profile or protein markers in EPs. Acute stimulation–derived EPs did not alter mitochondrial mass or cell count/viability. Further investigation into the effects of chronic contractile activity on the biophysical characteristics and cargo of skeletal muscle–specific EVs are warranted. Full article

Browning of adipocytes using herbal extracts is an attractive and realistic strategy for obesity treatment. Ephedra sinica Stapf (E. sinica) is an Asian traditional medicine known to activate brown adipocytes. To evaluate the effect of E. sinica (EEs) on the browning of white adipocytes, expression levels of browning markers, including uncoupling protein 1 (UCP1), were determined using qPCR, Western blot, and immunocytochemistry after mature mouse inguinal preadipocyte (mIPA) and human adipose-derived stem cells (hADSCs) were treated with EEs. In addition, mitochondrial activity was determined by analyzing MitoTracker staining, mtDNA copy number, and oxygen consumption rate (OCR). Treatment with EEs suppressed lipid accumulation and expression levels of adipogenic markers, including Pparg, during mIPA differentiation. In mature mIPA and hADSCs browning markers, including Ucp1, were up-regulated by EEs. In addition, EEs increased expression of mitochondrial genes, mtDNA copy number, and OCR. EEs showed a dual function: inhibiting adipogenesis in immature preadipocytes, and promoting thermogenesis via browning in mature white adipocytes. Therefore, E. sinica is a potential herb for regulating energy metabolism by inducing the browning process. Full article

The modes of interaction (e.g., mouse and touch) between maps and users affect the effectiveness and efficiency of transmitting cartographic information. Recent advances in eye tracking technology have made eye trackers lighter, cheaper and more accurate, broadening the potential to interact with maps via gaze. In this study, we focused exclusively on using gaze to choose map features (i.e., points, polylines and polygons) via the select operation, a fundamental action preceding other operations in map interactions. We adopted an approach based on the dwell time and buffer size to address the low spatial accuracy and Midas touch problem in gaze-based interactions and to determine the most suitable dwell time and buffer size for the gaze-based selection of map features. We conducted an experiment in which 38 participants completed a series of map feature selection tasks via gaze. We compared the participants’ performance (efficiency and accuracy) between different combinations of dwell times (200 ms, 600 ms and 1000 ms) and buffer sizes (point: 1°, 1.5°, and 2°; polyline: 0.5°, 0.7° and 1°). The results confirmed that a larger buffer size raised efficiency but reduced accuracy, whereas a longer dwell time lowered efficiency but enhanced accuracy. Specifically, we found that a 600 ms dwell time was more efficient in selecting map features than 200 ms and 1000 ms but was less accurate than 1000 ms. However, 600 ms was considered to be more appropriate than 1000 ms because a longer dwell time has a higher risk of causing visual fatigue. Therefore, 600 ms supports a better balance between accuracy and efficiency. Additionally, we found that buffer sizes of 1.5° and 0.7° were more efficient and more accurate than other sizes for selecting points and polylines, respectively. Our results provide important empirical evidence for choosing the most appropriate dwell times and buffer sizes for gaze-based map interactions. Full article