PIKfyve is an endosomal lipid kinase that synthesizes phosphatidylinositol 3,5-biphosphate from phosphatidylinositol 3-phsphate. Inhibition of PIKfyve activity leads to lysosomal enlargement and cytoplasmic vacuolation, attributed to impaired lysosomal fission processes and homeostasis. However, the precise molecular mechanisms underlying these effects remain a topic of debate. In this study, we present findings from PIKfyve-deficient zebrafish embryos, revealing enlarged macrophages with giant vacuoles reminiscent of lysosomal storage disorders. Treatment with mTOR inhibitors or effective knockout of mTOR partially reverses these abnormalities and extend the lifespan of mutant larvae. Further in vivo and in vitro mechanistic investigations provide evidence that PIKfyve activity is essential for mTOR shutdown during early zebrafish development and in cells cultured under serum-deprived conditions. These findings underscore the critical role of PIKfyve activity in regulating mTOR signaling and suggest potential therapeutic applications of PIKfyve inhibitors for the treatment of lysosomal storage disorders.

Canavan disease (CD) is a fatal hereditary neurological disorder caused by a mutation in the aspartoacylase (ASPA) gene and characterized by neurological signs and vacuolation in the central nervous system (CNS). The mutation inhibits the hydrolysis of N-acetyl-aspartate (NAA) resulting in accumulation of NAA in the CNS. A new Aspa-knockout rat was generated by transcription activator-like effector nuclease (TALEN) technology. Herein we describe the pathological and morphometrical findings in the brain and spinal cords of Aspa-knockout rats. Although Aspa-knockout rats did not show any neurological signs, vacuolation with swollen axons, hypomyelination, and activated swollen astrocytes were observed mainly in the brainstem reticular formation, ascending and descending motor neuron pathway, and in the olfactory tract. Morphometrical analysis revealed no obvious change in the number of neurons. These changes in the CNS are similar to human CD, suggesting that this animal model would be useful for further study of treatment and understanding the pathophysiology of human CD.

The Gram-negative bacterium Helicobacter pylori is a very successful pathogen, one of the most commonly identified causes of bacterial infections in humans worldwide. H. pylori produces several virulence factors that contribute to its persistence in the hostile host habitat and to its pathogenicity. The most extensively studied are cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA). VacA is present in almost all H. pylori strains. As a secreted multifunctional toxin, it assists bacterial colonization, survival, and proliferation during long-lasting infections. To exert its effect on gastric epithelium and other cell types, VacA undergoes several modifications and crosses multiple membrane barriers. Once inside the gastric epithelial cell, VacA disrupts many cellular-signaling pathways and processes, leading mainly to changes in the efflux of various ions, the depolarization of membrane potential, and perturbations in endocytic trafficking and mitochondrial function. The most notable effect of VacA is the formation of vacuole-like structures, which may lead to apoptosis. This review focuses on the processes involved in VacA secretion, processing, and entry into host cells, with a particular emphasis on the interaction of the mature toxin with host membranes and the formation of transmembrane pores.

The recently defined type of cell death ferroptosis has garnered significant attention as a potential new approach to cancer treatment owing to its more immunogenic nature when compared with apoptosis. Ferroptosis is characterized by the depletion of glutathione (GSH)/glutathione peroxidase-4 (GPx4) and iron-dependent lipid peroxidation. Diplacone (DP), a geranylated flavonoid compound found in Paulownia tomentosa fruit, has been identified to have anti-inflammatory and anti-radical activity. In this study, the potential anticancer activity of DP was explored against A549 human lung cancer cells. It was found that DP induced a form of cytotoxicity distinct from apoptosis, which was accompanied by extensive mitochondrial-derived cytoplasmic vacuoles. DP was also shown to increase mitochondrial Ca2+ influx, reactive oxygen species (ROS) production, and mitochondrial permeability transition (MPT) pore-opening. These changes led to decreases in mitochondrial membrane potential and DP-induced cell death. DP also induced lipid peroxidation and ATF3 expression, which are hallmarks of ferroptosis. The ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 were effective in counteracting the DP-mediated ferroptosis-related features. Our results could contribute to the use of DP as a ferroptosis-inducing agent, enabling studies focusing on the relationship between ferroptosis and the immunogenic cell death of cancer cells.

The spectrum of background, incidental, and experimentally induced lesions affecting NSG and NOG mice has been the subject of intense investigation. However, comprehensive studies focusing on the spontaneous neuropathological changes of these immunocompromised strains are lacking. This work describes the development of spontaneous early-onset neurodegeneration affecting both juvenile and adult NSG, NOG, and NXG mice. The study cohort consisted of 367 NSG mice of both sexes (including 33 NSG-SGM3), 61 NOG females (including 31 NOG-EXL), and 4 NXG females. These animals were primarily used for preclinical CAR T-cell testing, generation of humanized immune system chimeras, and/or tumor xenograft transplantation. Histopathology of brain and spinal cord and immunohistochemistry (IHC) for AIF-1, GFAP, CD34, and CD45 were performed. Neurodegenerative changes were observed in 57.6% of the examined mice (affected mice age range was 6-36 weeks). The lesions were characterized by foci of vacuolation with neuronal degeneration/death and gliosis distributed throughout the brainstem and spinal cord. IHC confirmed the development of gliosis, overexpression of CD34, and a neuroinflammatory component comprised of CD45-positive monocyte-derived macrophages. Lesions were significantly more frequent and severe in NOG mice. NSG males were considerably more affected than NSG females. Increased lesion frequency and severity in older animals were also identified. These findings suggest that NSG, NOG, and NXG mice are predisposed to the early development of identical neurodegenerative changes. While the cause of these lesions is currently unclear, potential associations with the genetic mutations shared by NSG, NOG, and NXG mice as well as unidentified viral infections are considered.

In this study, Hawthorn oligomic procyanidins extracts (HPOE) were evaluated for their anticancer activity on colorectal cancer. Our results showed that HPOE arrested HCT116 cells cycle at G2/M phase through P53-Cyclin B pathway and promoted apoptosis partly via mitochondrial (Caspase 9-Caspase 3) and death receptor (Caspase 8-Caspase 3) pathways. Meanwhile, it was found that HPOE aggravated HCT116 cells death through lysosomal vacuolation, which was verified by inhibitor/activator of P53-ILC3 signaling pathway. Taken together, HPOE exerted anticancer effects which lays the foundation for the development of functional foods for clinical colon cancer patients.

Glabridin, a polyphenolic flavonoid isolated from the root of the glycyrrhiza glabra, has been demonstrated to have anti-tumor properties in human malignancies. This study found that glabridin decreased the viability of human breast cancer MDA-MB-231 and MCF7 cells in a dose-dependent manner that was not involved in the caspase-3 cascade. Glabridin promoted the formation of extensive cytoplasmic vacuolation by increasing the expression of endoplasmic reticulum (ER) stress markers BiP, XBP1s, and CHOP. The transmission electron microscopy and fluorescence with the ER chaperon KDEL suggested that the vacuoles were derived from ER. Glabridin-induced vacuolation was blocked when protein synthesis was inhibited by cycloheximide, demonstrating that protein synthesis is crucial for this process. Furthermore, we determined that glabridin causes loss of mitochondrial membrane potential as well as the production of reactive oxygen species, both of which lead to mitochondrial dysfunction. These features are consistent with a kind of programmed cell death described as paraptosis. This work reports for the first time that glabridin could induce paraptosis-like cell death, which may give new therapeutic approaches for apoptosis-resistant breast cancers.

Conjugation to polyethylene glycol (PEG) is commonly used to enhance drug delivery and efficacy by extending the half-life of the drug molecule. This has important implications for reducing treatment burden in diseases that require chronic prophylaxis, such as hemophilia. Clearance of PEG molecules with high molecular weights (≥ 40 kDa) has been reported to cause cellular vacuolation in mammals. Rurioctocog alfa pegol (PEGylated recombinant coagulation factor VIII) contains a 20-kDa PEG. This study investigated the effects of exposure to 20-kDa PEG (10 μg/ml to 10 mg/ml) on the morphology and function of human monocyte-derived macrophages (MDMs) in vitro. Exposure to PEG for 24 hours was associated with significant vacuolation only at concentrations of 1 mg/ml or more, which far exceed the levels associated with clinically relevant doses of rurioctocog alfa pegol. Immunofluorescence staining of PEG was detected in the cytoplasm of MDMs, indicating uptake into the cells. No impairment of MDM phagocytic activity (ability to ingest fluorescently labeled Escherichia coli) was observed with 24-hour exposure to PEG, even at concentrations associated with significant vacuolation. Furthermore, PEG exposure did not have significant effects on cytokine secretion in resting or lipopolysaccharide-stimulated MDMs, or on the expression of cell surface markers in stimulated MDMs. Cell viability was not affected by 24-hour exposure to PEG. In conclusion, vacuolation of human MDMs after exposure to 20-kDa PEG only occurred with PEG concentrations far in excess of those equivalent to clinically relevant doses of rurioctocog alfa pegol and did not affect MDM viability or functionality. Together, these results support the concept that PEG-mediated vacuolation is an adaptive cellular response rather than a toxic effect.

Jintrolong® is a long-acting PEGylated recombinant human growth hormone (PEG-rhGH) developed for weekly injection in patients with pediatric growth hormone deficiency (PGHD). Although PEG modification of therapeutic proteins is generally considered safe, concerns persist about the potential for adverse vacuolation in tissues with long-term exposure to PEG-included therapies, particularly in children. We assessed the safety of Jintrolong® in cynomolgus monkeys with an examination of vacuolation in the brain choroid plexus (CP) and reported long-term clinical safety data obtained from children with PGHD. The toxicity of Jintrolong® was assessed following the 52-week administration with doses at 0.3, 1, or 3 mg/kg/week. The levels of vacuolation of CP in animals were dose-dependent and at least partially reversible after a 104- or 157-week recovery period. Vacuolation in the CP epithelium did not lead to obvious subcellular structural or cell functional abnormalities. Compared with the clinical dose of 0.2 mg/kg/week Jintrolong® in PGHD patients, exposure in monkeys under NOAEL 3 mg/kg/week exhibited safety margins greater than 120.5, the predicted minimum dose to induce vacuolation in monkeys is equivalent to 1.29 mg/kg/week in humans, which is 6.45-fold higher than the clinical dose. The safety data acquired in clinical trials for Jintrolong® were also analyzed, which included phase III (360 patients), phase IV (3,000 patients) of 26-week treatment, and a follow-up study with treatment lasting for 3 years. There was no statistically significant difference in the incidence of adverse reactions between the Jintrolong® group and the daily rhGH control group (no PEG), and no new adverse effects (AE) were observed in the Jintrolong® group at the clinical therapeutic dose of 0.2 mg/kg/week.

β2 integrins are critical for neutrophil firm adhesion, trans-endothelial migration, and the recruitment to the inflamed tissue. Autophagy is implicated in cell migration and tumor metastasis through facilitating the turnover of β1 integrins; however, whether autophagy is able to control neutrophil migration by promoting the degradation of β2 integrins is unexplored. Here, we show that high blood levels of palmitic acid (PA) strongly triggered neutrophil autophagy activation, leading to adhesion deficiency in dairy cows with fatty liver. The three neutrophil granule subtypes, namely, azurophil granules (AGs), specific granules (SGs), and gelatinase granules (GGs), were engulfed by the autophagosomes for degradation, resulting in an increased vacuolation in fatty liver dairy cow neutrophils. Importantly, the adhesion-associated molecules CD11b and CD18 distributed on AGs, SGs, and GGs were degraded with the three granule subtypes by autophagy. Moreover, FGA, Hsc70, and TRIM21 mediated the degradation of cytosolic oxidized-ubiquitinated CD11b and CD18. Collectively, our results demonstrate that high blood PA triggers neutrophil autophagy-dependent vacuolation and granule-dependent adhesion deficiency, decreasing neutrophil mobility, and impairing the innate immune system of dairy cow with fatty liver. This theory extends the category of autophagy in maintaining granule homeostasis and provides a novel strategy to improve the immune of dairy cows with metabolic disease.