A high dose KRP203 induces cytoplasmic vacuoles associated with altered phosphoinositide segregation and endosome expansion.

In animal cells, vacuoles are absent, but can be induced by diseases and drugs. While phosphoinositides are critical for membrane trafficking, their role in the formation of these vacuoles remains unclear. The immunosuppressive KRP203/Mocravimod, which antagonizes sphingosine-1-phosphate receptors, has been identified as having novel multimodal activity against phosphoinositide kinases. However, the impact of this novel KRP203 activity is unknown. Here, we show that KRP203 disrupts the spatial organization of phosphoinositides and induces extensive vacuolization in tumor cells and immortalized fibroblasts. The KRP203-induced vacuoles are primarily from endosomes, and augmented by inhibition of PIKFYVE and VPS34. Conversely, overexpression of PTEN decreased KRP203-induced vacuole formation. Furthermore, V-ATPase inhibition completely blunted KRP203-induced vacuolization, pointing to a critical requirement of the endosomal maturation process. Importantly, nearly a half of KRP203-induced vacuoles are significantly decorated with PI4P, a phosphoinositide typically enriched at the plasma membrane and Golgi. These results suggest a model that noncanonical spatial reorganization of phosphoinositides by KRP203 alters the endosomal maturation process, leading to vacuolization. Taken together, this study reveals a previously unrecognized bioactivity of KRP203 as a vacuole-inducing agent and its unique mechanism of phosphoinositide modulation, providing a new insight of phosphoinositide regulation into vacuolization-associated diseases and their molecular pathologies.

The iron chelator deferriferrichrysin induces paraptosis via extracellular signal-related kinase activation in cancer cells.

Cancer cells generally exhibit increased iron uptake, which contributes to their abnormal growth and metastatic ability. Iron chelators have thus recently attracted attention as potential anticancer agents. Here, we show that deferriferrichrysin (Dfcy), a natural product from Aspergillus oryzae acts as an iron chelator to induce paraptosis (a programmed cell death pathway characterized by ER dilation) in MCF-7 human breast cancer cells and H1299 human lung cancer cells. We first examined the anticancer efficacy of Dfcy in cancer cells and found that Dfcy induced ER dilation and reduced the number of viable cells. Extracellular signal-related kinase (ERK) was activated by Dfcy treatment, and the MEK inhibitor U0126, a small molecule commonly used to inhibit ERK activity, prevented the increase in ER dilation in Dfcy-treated cells. Concomitantly, the decrease in the number of viable cells upon treatment with Dfcy was attenuated by U0126. Taken together, these results demonstrate that the iron chelator Dfcy exhibits anticancer effects via induction of ERK-dependent paraptosis.

Triptolide exposure triggers testicular vacuolization injury by disrupting the Sertoli cell junction and cytoskeletal organization via the AKT/mTOR signaling pathway.

BACKGROUND: Despite the known reproductive toxicity induced by triptolide (TP) exposure, the regulatory mechanism underlying testicular vacuolization injury caused by TP remains largely obscure. METHODS: Male mice were subjected to TP at doses of 15, 30, and 60 µg/kg for 35 consecutive days. Primary Sertoli cells were isolated from 20-day-old rat testes and exposed to TP at concentrations of 0, 40, 80, 160, 320, and 640 nM. A Biotin tracer assay was conducted to assess the integrity of the blood-testis barrier (BTB). Transepithelial electrical resistance (TER) assays were employed to investigate BTB function in primary Sertoli cells. Histological structures of the testes and epididymides were stained with hematoxylin and eosin (H&E). The expression and localization of relevant proteins or pathways were assessed through Western blotting or immunofluorescence staining. RESULTS: TP exposure led to dose-dependent testicular injuries, characterized by a decreased organ coefficient, reduced sperm concentration, and the formation of vacuolization damage. Furthermore, TP exposure disrupted BTB integrity by reducing the expression levels of tight junction (TJ) proteins in the testes without affecting basal ectoplasmic specialization (basal ES) proteins. Through the TER assay, we identified that a TP concentration of 160 nM was optimal for elucidating BTB function in primary Sertoli cells, correlating with reductions in TJ protein expression. Moreover, TP exposure induced changes in the distribution of the BTB and cytoskeleton-associated proteins in primary Sertoli cells. By activating the AKT/mTOR signaling pathway, TP exposure disturbed the balance between mTORC1 and mTORC2, ultimately compromising BTB integrity in Sertoli cells. CONCLUSION: This investigation sheds light on the impacts of TP exposure on testes, elucidating the mechanism by which TP exposure leads to testicular vacuolization injury and offering valuable insights into comprehending the toxic effects of TP exposure on testes.

Comparative effects of chemical and green zinc oxide nanoparticles in caprine testis: ultrastructural and steroidogenic enzyme analysis.

Recent advancements in nanotechnology has opened up enormous possibilities in diverse sectors such as industries, agriculture, environmental remediation, electronics, medicine and varied industries. Among metal oxide nanoparticles zinc oxide nanoparticles has gained considerable attention due to their fascinating physiochemical properties. Rapid growth in the use of zinc oxide nanoparticles (ZnONPs) in daily household products, food and feed additives, biological products, medicine, as antimicrobial agents, electronics and agriculture, creates serious toxic potential risks of these engineered nanoparticles on living organisms. The aim of present study was to assess the effects of synthesized chemical ZnONPs and green ZnONPs on testicular tissue of Capra hircus (goat) in vitro. The reproductive stress was analyzed by ultrastructural damage, change in frequency of apoptotic cells and alteration in steroidogenic enzyme activity. The transmission electron micrographs of testicular cells after treatment with chemical and green ZnONPs at three doses (10 µg/ml, 20 µg/ml and 30 µg/ml) for exposure duration 4 h and 8 h illustrated that chemical nanoparticles induced more alterations, identified as ruptured nuclear membrane, condensation and margination of chromatin material in somatic cells and germ cells in the seminiferous tubules, presence of apoptotic bodies in nucleus of spermatocytes and spermatids, reduction in number of cell organelles, vacuolization and hyalinization of cytoplasm. Maximum damage was observed after treatment of testicular tissues with 30 µg/ml of chemical ZnONPs for 8 h exposure duration. However, the green ZnONPs were found to be less toxic as evidenced by few apoptotic characteristics in testicular cells. The results of fluorescence assay by acridine orange staining showed significant increase in the percentage of apoptotic cells in chemical treated groups as compared to green and control groups. Decreased enzyme activity of 3ß-Hydroxysteroid dehydrogenase and 17ß-Hydroxysteroid dehydrogenase was assayed in chemical ZnONPs than green ZnONPs treated groups. Our results confirm that chemical ZnONPs are significantly more toxic in comparison to green ZnONPs and adversely affects the male fertility.

The pyrazole derivative of usnic acid inhibits the proliferation of pancreatic cancer cells in vitro and in vivo.

BACKGROUND: Pancreatic cancer is one of the leading causes of cancer death in Western societies. Its late diagnosis and resistance to chemotherapies result in a high mortality rate; thus, the development of more effective therapies for the treatment of pancreatic cancer is strongly warranted. Usnic acid (UA) is a secondary metabolite of lichens that shows modest antiproliferative activity toward cancer cells. Recently, we reported the synthesis of a UA pyrazole derivative, named 5, which was more active than the parent compound toward cervical cancer cells. Here, its anticancer potential has been evaluated in detail in other cancer cells, particularly pancreatic cancer cells. METHODS: The impact of UA and derivative 5 on cell viability, morphology, cell cycle, and death was assessed using the MTT test, electron microscopy, flow cytometry, and immunoblotting, respectively. The calcium ions level was detected fluorometrically. In vivo, the anticancer activity of 5 was evaluated in a murine xenograft model. RESULTS: Derivative 5 inhibited the viability of different cancer cells. Noncancerous cells were less sensitive. It induced the release of calcium ions from the endoplasmic reticulum (ER) and ER stress, which was manifested by cell vacuolization. It was accompanied by G0/G1 cell cycle arrest and cell death of pancreatic cancer cells. When applied to nude mice with xenografted pancreatic cancer cells, 5 inhibited tumor growth, with no signs of kidney or liver toxicity. CONCLUSIONS: UA derivative 5 is superior to UA inhibiting the growth and proliferation of pancreatic cancer cells. ER stress exaggeration is a mechanism underlying the activity of derivative 5.

Neuropathological report of propionic acidemia.

Propionic acidemia (PA) is an autosomal recessive inheritable metabolic disease caused by mutations in the propionyl CoA carboxylase gene (PCC) that affects multiple systems of the human body. Here, we report neuropathological findings of a PA patient. The patient was a male infant who presented with increasing lethargy and poor feeding from four days postpartum. He gradually became comatose and died from complications after liver transplantation at three months old. The results of laboratory examination were consistent with PA, and genetic analysis revealed compound heterozygous mutations in the gene for PCC subunit beta: c.838dupC (rs769968548) and c.1127G>T (rs142982097). Brain-restricted autopsy was performed 23 h after his death, and the neuropathological examination revealed distinct astrocytosis, oligodendrocytic loss, neuronal loss, and demyelination across the brainstem, motor cortex, basal ganglia, and thalamus. Spongiosis, vacuolization, and the appearance of Alzheimer type II astrocytes and activated microglia were observed as well. This is the first brain autopsy report of PA with a clear genetic cause.

Cellular basis of ClC-2 Cl- channel-related brain and testis pathologies.

The ClC-2 chloride channel is expressed in the plasma membrane of almost all mammalian cells. Mutations that cause the loss of ClC-2 function lead to retinal and testicular degeneration and leukodystrophy, whereas gain-of-function mutations cause hyperaldosteronism. Leukodystrophy is also observed with a loss of GlialCAM, a cell adhesion molecule that binds to ClC-2 in glia. GlialCAM changes the localization of ClC-2 and opens the channel by altering its gating. We now used cell type-specific deletion of ClC-2 in mice to show that retinal and testicular degeneration depend on a loss of ClC-2 in retinal pigment epithelial cells and Sertoli cells, respectively, whereas leukodystrophy was fully developed only when ClC-2 was disrupted in both astrocytes and oligodendrocytes. The leukodystrophy of Glialcam-/- mice could not be rescued by crosses with Clcn2op/op mice in which a mutation mimics the "opening" of ClC-2 by GlialCAM. These data indicate that GlialCAM-induced changes in biophysical properties of ClC-2 are irrelevant for GLIALCAM-related leukodystrophy. Taken together, our findings suggest that the pathology caused by Clcn2 disruption results from disturbed extracellular ion homeostasis and identifies the cells involved in this process.

Methuosis Contributes to Jaspine-B-Induced Cell Death.

Methuosis is a type of programmed cell death in which the cytoplasm is occupied by fluid-filled vacuoles that originate from macropinosomes (cytoplasmic vacuolation). A few molecules have been reported to behave as methuosis inducers in cancer cell lines. Jaspine B (JB) is a natural anhydrous sphingolipid (SL) derivative reported to induce cytoplasmic vacuolation and cytotoxicity in several cancer cell lines. Here, we have investigated the mechanism and signalling pathways involved in the cytotoxicity induced by the natural sphingolipid Jaspine B (JB) in lung adenocarcinoma A549 cells, which harbor the G12S K-Ras mutant. The effect of JB on inducing cytoplasmic vacuolation and modifying cell viability was determined in A549 cells, as well as in mouse embryonic fibroblasts (MEF) lacking either the autophagy-related gene ATG5 or BAX/BAK genes. Apoptosis was analyzed by flow cytometry after annexin V/propidium iodide staining, in the presence and absence of z-VAD. Autophagy was monitored by LC3-II/GFP-LC3-II analysis, and autophagic flux experiments using protease inhibitors. Phase contrast, confocal, and transmission electron microscopy were used to monitor cytoplasmic vacuolation and the uptake of Lucifer yellow to assess macropinocyosis. We present evidence that cytoplasmic vacuolation and methuosis are involved in Jaspine B cytotoxicity over A549 cells and that activation of 5' AMP-activated protein kinase (AMPK) could be involved in Jaspine-B-induced vacuolation, independently of the phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin complex 1 (PI3K/Akt/mTORC1) axis.

Maduramicin induces cardiotoxicity via Rac1 signaling-independent methuosis in H9c2 cells.

Maduramicin frequently induces severe cardiotoxicity in target and nontarget animals in clinic. Apoptotic and non-apoptotic cell death mediate its cardiotoxicity; however, the underlying non-apoptotic cell death induced by maduramicin remains unclear. In current study, a recently described non-apoptotic cell death "methuosis" caused by maduramicin was defined in mammalian cells. Rat myocardial cell H9c2 was used as an in vitro model, showing excessively cytoplasmic vacuolization upon maduramicin (0.0625-5 µg/mL) exposure for 24 h. Maduramicin-induced reversible cytoplasmic vacuolization of H9c2 cells in a time- and concentration-dependent manner. The vacuoles induced by maduramicin were phase lucent with single membrane and were not derived from the swelling of organelles such as mitochondria, endoplasmic reticulum, lysosome, and Golgi apparatus. Furthermore, maduramicin-induced cytoplasmic vacuoles are generated from micropinocytosis, which was demonstrated by internalization of extracellular fluid-phase marker Dextran-Alexa Fluor 488 into H9c2 cells. Intriguingly, these cytoplasmic vacuoles acquired some characteristics of late endosomes and lysosomes rather than early endosomes and autophagosomes. Vacuolar H+ -ATPase inhibitor bafilomycin A1 efficiently prevented the generation of cytoplasmic vacuoles and decreased the cytotoxicity of H9c2 cells triggered by maduramicin. Mechanism studying indicated that maduramicin activated H-Ras-Rac1 signaling pathway at both mRNA and protein levels. However, the pharmacological inhibition and siRNA knockdown of Rac1 rescued maduramicin-induced cytotoxicity of H9c2 cells but did not alleviate cytoplasmic vacuolization. Based on these findings, maduramicin induces methuosis in H9c2 cells via Rac-1 signaling-independent seriously cytoplasmic vacuolization.

Tissue-Specific Proteome and Subcellular Microscopic Analyses Reveal the Effect of High Salt Concentration on Actin Cytoskeleton and Vacuolization in Aleurone Cells during Early Germination of Barley.

Cereal grain germination provides the basis for crop production and requires a tissue-specific interplay between the embryo and endosperm during heterotrophic germination involving signalling, protein secretion, and nutrient uptake until autotrophic growth is possible. High salt concentrations in soil are one of the most severe constraints limiting the germination of crop plants, affecting the metabolism and redox status within the tissues of germinating seed. However, little is known about the effect of salt on seed storage protein mobilization, the endomembrane system, and protein trafficking within and between these tissues. Here, we used mass spectrometry analyses to investigate the protein dynamics of the embryo and endosperm of barley (Hordeum vulgare, L.) at five different early points during germination (0, 12, 24, 48, and 72 h after imbibition) in germinated grains subjected to salt stress. The expression of proteins in the embryo as well as in the endosperm was temporally regulated. Seed storage proteins (SSPs), peptidases, and starch-digesting enzymes were affected by salt. Additionally, microscopic analyses revealed an altered assembly of actin bundles and morphology of protein storage vacuoles (PSVs) in the aleurone layer. Our results suggest that besides the salt-induced protein expression, intracellular trafficking and actin cytoskeleton assembly are responsible for germination delay under salt stress conditions.

Cyclometalated Iridium(III) Complex-Cationic Peptide Hybrids Trigger Paraptosis in Cancer Cells via an Intracellular Ca2+ Overload from the Endoplasmic Reticulum and a Decrease in Mitochondrial Membrane Potential.

In our previous paper, we reported that amphiphilic Ir complex-peptide hybrids (IPHs) containing basic peptides such as KK(K)GG (K: lysine, G: glycine) (e.g., ASb-2) exhibited potent anticancer activity against Jurkat cells, with the dead cells showing a strong green emission. Our initial mechanistic studies of this cell death suggest that IPHs would bind to the calcium (Ca2+)-calmodulin (CaM) complex and induce an overload of intracellular Ca2+, resulting in the induction of non-apoptotic programmed cell death. In this work, we conduct a detailed mechanistic study of cell death induced by ASb-2, a typical example of IPHs, and describe how ASb-2 induces paraptotic programmed cell death in a manner similar to that of celastrol, a naturally occurring triterpenoid that is known to function as a paraptosis inducer in cancer cells. It is suggested that ASb-2 (50 µM) induces ER stress and decreases the mitochondrial membrane potential (ΔΨm), thus triggering intracellular signaling pathways and resulting in cytoplasmic vacuolization in Jurkat cells (which is a typical phenomenon of paraptosis), while the change in ΔΨm values is negligibly induced by celastrol and curcumin. Other experimental data imply that both ASb-2 and celastrol induce paraptotic cell death in Jurkat cells, but this induction occurs via different signaling pathways.

Endoplasmic reticulum: a focal point of Zika virus infection.

Zika virus (ZIKV) belongs to the Flavivirus genus of the Flaviviridae family. It is an arbovirus that can cause congenital abnormalities and is sexually transmissible. A series of outbreaks accompanied by unexpected severe clinical complications have captured medical attention to further characterize the clinical features of congenital ZIKV syndrome and its underlying pathophysiological mechanisms. Endoplasmic reticulum (ER) and ER-related proteins are essential in ZIKV genome replication. This review highlights the subcellular localization of ZIKV to the ER and ZIKV modulation on the architecture of the ER. This review also discusses ZIKV interaction with ER proteins such as signal peptidase complex subunit 1 (SPCS1), ER membrane complex (EMC) subunits, and ER translocon for viral replication. Furthermore, the review covers several important resulting effects of ZIKV infection to the ER and cellular processes including ER stress, reticulophagy, and paraptosis-like death. Pharmacological targeting of ZIKV-affected ER-resident proteins and ER-associated components demonstrate promising signs of combating ZIKV infection and rescuing host organisms from severe neurologic sequelae.

Leukodystrophies and genetic leukoencephalopathies in children.

Leukodystrophies and genetic leukoencephalopathies are a large group of genetic disorders affecting central nervous system white matter. They can begin at any age, however this study focuses on disorders beginning in childhood and adolescence. We discuss the recent definitions, classifications, and classic syndromes, as well as genetic progress in the field through the identification of new genes and several new genetic syndromes.

Cerebral hypomyelination associated with biallelic variants of FIG4.

The lipid phosphatase gene FIG4 is responsible for Yunis-Varón syndrome and Charcot-Marie-Tooth disease Type 4J, a peripheral neuropathy. We now describe four families with FIG4 variants and prominent abnormalities of central nervous system (CNS) white matter (leukoencephalopathy), with onset in early childhood, ranging from severe hypomyelination to mild undermyelination, in addition to peripheral neuropathy. Affected individuals inherited biallelic FIG4 variants from heterozygous parents. Cultured fibroblasts exhibit enlarged vacuoles characteristic of FIG4 dysfunction. Two unrelated families segregate the same G > A variant in the +1 position of intron 21 in the homozygous state in one family and compound heterozygous in the other. This mutation in the splice donor site of exon 21 results in read-through from exon 20 into intron 20 and truncation of the final 115 C-terminal amino acids of FIG4, with retention of partial function. The observed CNS white matter disorder in these families is consistent with the myelination defects in the FIG4 null mouse and the known role of FIG4 in oligodendrocyte maturation. The families described here the expanded clinical spectrum of FIG4 deficiency to include leukoencephalopathy.

Pseudomonas sp. COW3 Produces New Bananamide-Type Cyclic Lipopeptides with Antimicrobial Activity against Pythium myriotylum and Pyricularia oryzae.

Pseudomonas species are metabolically robust, with capacity to produce secondary metabolites including cyclic lipopeptides (CLPs). Herein we conducted a chemical analysis of a crude CLP extract from the cocoyam rhizosphere-derived biocontrol strain Pseudomonas sp. COW3. We performed in silico analyses on its whole genome, and conducted in vitro antagonistic assay using the strain and purified CLPs. Via LC-MS and NMR, we elucidated the structures of four novel members of the bananamide group, named bananamides D-G. Besides variability in fatty acid length, bananamides D-G differ from previously described bananamides A-C and MD-0066 by the presence of a serine and aspartic acid at position 6 and 2, respectively. In addition, bananamide G has valine instead of isoleucine at position 8. Kendrick mass defect (KMD) allowed the assignment of molecular formulae to bananamides D and E. We unraveled a non-ribosomal peptide synthetase cluster banA, banB and banC which encodes the novel bananamide derivatives. Furthermore, COW3 displayed antagonistic activity and mycophagy against Pythium myriotylum, while it mainly showed mycophagy on Pyricularia oryzae. Purified bananamides D-G inhibited the growth of P. myriotylum and P. oryzae and caused hyphal distortion. Our study shows the complementarity of chemical analyses and genome mining in the discovery and elucidation of novel CLPs. In addition, structurally diverse bananamides differ in their antimicrobial activity.

Automated Bright Field Segmentation of Cells and Vacuoles Using Image Processing Technique.

Understanding the mechanisms and other variants of programmed cell death will help provide deeper insight into various disease processes. Although complex procedures are required to distinguish each type of cell death, the formation of vacuoles is one of the important features in some process of cell death under different conditions. Thus, monitoring and counting the number of vacuoles and the ratio of cells with vacuoles is a commonly used method to indicate and quantify the efficacy of the therapy. Several studies have shown that image processing can provide a quick, convenient and precise mean of performing cell detection. Hence, this study uses an image processing technique to detect and quantify vacuolated cells without the need for dyes. The system both counts the number of vacuolated cells and determines the ratio of cells with vacuoles. The performance of the proposed image processing system was evaluated using 38 images. It has been shown that a strong correlation exists between the automated counts and the manual counts. Furthermore, the absolute percentage errors between automated counts and manual counts for cell detection and vacuolated cell detection using data pooled from all images are 3.61 and 3.33%, respectively. A user-friendly graphical user interface (GUI) is also developed and freely available for download, providing researchers in biomedicine with a more convenient instrument for vacuolization analysis.

Spores potentially dispersed to longer distances are more tolerant to ultraviolet radiation: A case study in the moss genus Orthotrichum.

PREMISE OF THE STUDY: Ultraviolet (UV) radiation influences the viability of algal spores and seed-plant pollen depending on the species, the dose, and the wavelength. In bryophytes, one of the dominant groups of plants in many habitats, UV radiation could determine their spore dispersal strategy, and such data are critical for reconstructing the ancestral state in plants and for determining the distribution range and persistence of bryophyte species. METHODS: Spores of four bryophyte species of the moss genus Orthotrichum that were either hygrochastic or xerochastic (spores dispersed under wet or dry conditions, respectively) were exposed to realistic doses of UV radiation under laboratory conditions. Spore viability was evaluated through germination experiments and, for the first time in bryophytes, ultrastructural observations. Given that the UV-B doses used were relatively higher than the UV-A doses, the UV effect was probably due more to UV-B than UV-A wavelengths. KEY RESULTS: All four species reduced their spore germination capacity in a UV dose-dependent manner, concomitantly increasing spore ultrastructural damage (cytoplasmic and plastid alterations). Most spores eventually died when exposed to the highest UV dose. Interestingly, spores of hygrochastic species were much more UV-sensitive than those of xerochastic species. CONCLUSIONS: UV tolerance determines moss spore viability, as indicated by germination capacity and ultrastructural damage, and differs between spores of species with different dispersal strategies. Specifically, the higher UV tolerance of xerochastic spores may enable them to be dispersed to longer distances than hygrochastic spores, thus extending more efficiently the distribution range of the corresponding species.

Desipramine-induced lysosomal vacuolization is independent of autophagy.

Desipramine, a commonly used antidepressant drug, induced cytosolic vacuolization in L929 cells. The level of LC3-II was elevated and that of p62 was reduced in desipramine-treated L929 cells, indicating the induction of autophagy by desipramine. Surprisingly, massive vacuolization was observed in desipramine-treated L929 cells in the presence of LY294002, an inhibitor of autophagy. On the other hand, bafilomycin A1, an inhibitor of vacuolar type H+ ATPase, almost completely inhibited vacuolization in desipramine- or desipramine/LY294002-treated L929 cells. Furthermore, desipramine-induced vacuolization was observed in autophagy-deficient Atg7-/- mouse embryonic fibroblasts (MEFs) as well as wild-type Atg7+/+ MEFs. These results demonstrate that desipramine-induced lysosomal vacuolization is independent of autophagy.

Bone marrow features in Pearson syndrome with neonatal onset: A case report and review of the literature.

Pearson syndrome (PS) is a rare mitochondrial disorder that usually presents with transfusion-dependent macrocytic anemia, exocrine pancreatic dysfunction, and lactic acidosis. Typical bone marrow (BM) features are vacuolization in hematopoietic progenitors, hypocellularity, and ringed sideroblasts. At the neonatal age, PS may have a variable clinical onset. Moreover, there is little information about BM features at this age and the timing of their presentation. We report a neonatal case of PS that presented with refractory anemia and atypical BM features. We reviewed the BM findings in neonatal-onset PS cases to stress the importance and limitations of BM evaluation at this age.

Neuronal Vacuolization in Feline Panleukopenia Virus Infection.

Feline panleukopenia virus (FPV) infections are typically associated with anorexia, vomiting, diarrhea, neutropenia, and lymphopenia. In cases of late prenatal or early neonatal infections, cerebellar hypoplasia is reported in kittens. In addition, single cases of encephalitis are described. FPV replication was recently identified in neurons, although it is mainly found in cells with high mitotic activity. A female cat, 2 months old, was submitted to necropsy after it died with neurologic deficits. Besides typical FPV intestinal tract changes, multifocal, randomly distributed intracytoplasmic vacuoles within neurons of the thoracic spinal cord were found histologically. Next-generation sequencing identified FPV-specific sequences within the central nervous system. FPV antigen was detected within central nervous system cells, including the vacuolated neurons, via immunohistochemistry. In situ hybridization confirmed the presence of FPV DNA within the vacuolated neurons. Thus, FPV should be considered a cause for neuronal vacuolization in cats presenting with ataxia.