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Nature Communications Jun 2024Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require...
Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require a complex choreography of numerous factors. The endosomal sorting complex required for transport (ESCRT) is implicated in the final step of autophagosomal maturation by sealing of the phagophore membrane. ESCRT-III components were shown to mediate membrane scission by forming filaments that interact with cellular membranes. However, the molecular mechanisms underlying the recruitment of ESCRTs to non-endosomal membranes remain largely unknown. Here we focus on the ESCRT-associated protein ALG2-interacting protein X (ALIX) and identify Ca-dependent lipid binding protein 1 (CaLB1) as its interactor. Our findings demonstrate that CaLB1 interacts with AUTOPHAGY8 (ATG8) and PI(3)P, a phospholipid found in autophagosomal membranes. Moreover, CaLB1 and ALIX localize with ATG8 on autophagosomes upon salt treatment and assemble together into condensates. The depletion of CaLB1 impacts the maturation of salt-induced autophagosomes and leads to reduced delivery of autophagosomes to the vacuole. Here, we propose a crucial role of CaLB1 in augmenting phase separation of ALIX, facilitating the recruitment of ESCRT-III to the site of phagophore closure thereby ensuring efficient maturation of autophagosomes.
Topics: Arabidopsis; Autophagosomes; Endosomal Sorting Complexes Required for Transport; Arabidopsis Proteins; Calcium-Binding Proteins; Autophagy; Phosphatidylinositol Phosphates; Autophagy-Related Protein 8 Family; Vacuoles; Phase Separation
PubMed: 38898014
DOI: 10.1038/s41467-024-49485-6 -
Acta Neuropathologica Jun 2024TAR DNA-binding protein 43 (TDP-43) is an RNA binding protein found within ribonucleoprotein granules tethered to lysosomes via annexin A11. TDP-43 protein forms...
TAR DNA-binding protein 43 (TDP-43) is an RNA binding protein found within ribonucleoprotein granules tethered to lysosomes via annexin A11. TDP-43 protein forms inclusions in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and limbic predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC). Annexin A11 is also known to form aggregates in ALS cases with pathogenic variants in ANXA11. Annexin A11 aggregation has not been described in sporadic ALS, FTLD-TDP or LATE-NC cases. To explore the relationship between TDP-43 and annexin A11, genetic analysis of 822 autopsy cases was performed to identify rare ANXA11 variants. In addition, an immunohistochemical study of 368 autopsy cases was performed to identify annexin A11 aggregates. Insoluble annexin A11 aggregates which colocalize with TDP-43 inclusions were present in all FTLD-TDP Type C cases. Annexin A11 inclusions were also seen in a small proportion (3-6%) of sporadic and genetic forms of FTLD-TDP types A and B, ALS, and LATE-NC. In addition, we confirm the comingling of annexin A11 and TDP-43 aggregates in an ALS case with the pathogenic ANXA11 p.G38R variant. Finally, we found abundant annexin A11 inclusions as the primary pathologic finding in a case of progressive supranuclear palsy-like frontotemporal dementia with prominent striatal vacuolization due to a novel variant, ANXA11 p.P75S. By immunoblot, FTLD-TDP with annexinopathy and ANXA11 variant cases show accumulation of insoluble ANXA11 including a truncated fragment. These results indicate that annexin A11 forms a diverse and heterogeneous range of aggregates in both sporadic and genetic forms of TDP-43 proteinopathies. In addition, the finding of a primary vacuolar annexinopathy due to ANXA11 p.P75S suggests that annexin A11 aggregation is sufficient to cause neurodegeneration.
Topics: Humans; Aged; Annexins; Female; Male; DNA-Binding Proteins; Frontotemporal Lobar Degeneration; Middle Aged; Aged, 80 and over; TDP-43 Proteinopathies; Neurodegenerative Diseases; Amyotrophic Lateral Sclerosis; Inclusion Bodies; Brain; Protein Aggregation, Pathological
PubMed: 38896345
DOI: 10.1007/s00401-024-02753-7 -
BioRxiv : the Preprint Server For... Jun 2024is a Gram-negative bacterium found in a wide variety of water and land environments and organisms. It has been isolated as part of the gut microbiome of animals and...
is a Gram-negative bacterium found in a wide variety of water and land environments and organisms. It has been isolated as part of the gut microbiome of animals and insects, as well as from stool samples of patients with diarrhea. Specific strains encode gene homologs of virulence factors found in other pathogenic members of the same Enterobacterales order, such as serovar Typhimurium and Whether these genes are also pathogenic determinants in is not known. Here we have used 205/92, a clinical isolate, with and infection models to investigate -host interactions at the cellular level. Our particular focus was the role of two type III secretion systems (T3SS) belonging to the Inv-Mxi/Spa family. T3SS is widespread in spp. and encoded on the chromosome. T3SS is encoded on a large plasmid that is present in a subset of strains, which are primarily isolates from diarrheal patients. Using a combination of electron and fluorescence microscopy and gentamicin protection assays we show that 205/92 is internalized into eukaryotic cells, rapidly lyses its internalization vacuole and proliferates in the cytosol. This triggers caspase-4 dependent inflammasome responses in gut epithelial cells. The requirement for the T3SS in entry, vacuole lysis and cytosolic proliferation is host-cell type specific, playing a more prominent role in human intestinal epithelial cells as compared to macrophages. In a bovine ligated intestinal loop model, colonizes the intestinal mucosa, inducing mild epithelial damage with negligible fluid accumulation. No overt role for T3SS or T3SS was seen in the calf infection model. However, T3SS was required for the rapid killing of . We propose that the acquisition of two T3SS by horizontal gene transfer has allowed to diversify its host range, from a highly virulent pathogen of insects to an opportunistic gastrointestinal pathogen of animals.
PubMed: 38895369
DOI: 10.1101/2024.06.07.595826 -
BioRxiv : the Preprint Server For... Jun 2024Anthracyclines, such as doxorubicin, are important anti-cancer therapies but are associated with arterial injury. Histopathological insights have been limited to small...
BACKGROUND
Anthracyclines, such as doxorubicin, are important anti-cancer therapies but are associated with arterial injury. Histopathological insights have been limited to small animal models and the role of inflammation in the arterial toxic effects of anthracycline is unclear in humans. Our aims were: 1) To evaluate aortic media fibrosis and injury in non-human primates treated with anthracyclines; 2) To assess the effect of anthracycline on aortic inflammation in patients treated for lymphoma.
METHODS
1) African Green monkeys (AGM) received doxorubicin (30-60 mg/m/biweekly IV, cumulative dose: 240 mg/m). Blinded histopathologic analyses of collagen deposition and cell vacuolization in the ascending aorta were performed 15 weeks after the last doxorubicin dose and compared to 5 age- and gender-matched healthy, untreated AGMs. 2) Analysis of the thoracic aorta of patients with diffuse large B-cell lymphoma (DLBCL), at baseline and after doxorubicin exposure, was performed using F-fluorodeoxyglucose (F-FDG) positron emission tomography/computed tomography (PET/CT) in this observational study. The primary outcome was change in maximal tissue-to-background ratio (TBRmax) of the thoracic aorta from baseline to their end-of-treatment clinical PET/CT.
RESULTS
In AGMs, doxorubicin exposure was associated with greater aortic fibrosis (collagen deposition: doxorubicin cohort 6.23±0.88% vs. controls 4.67±0.54%; p=0.01) and increased intracellular vacuolization (doxorubicin 66.3 ± 10.1 vs controls 11.5 ± 4.2 vacuoles/field, p<0.0001) than untreated controls.In 101 patients with DLBCL, there was no change in aortic TBRmax after anthracycline exposure (pre-doxorubicin TBRmax 1.46±0.16 vs post-doxorubicin TBRmax 1.44±0.14, p=0.14). The absence of change in TBRmax was consistent across all univariate analyses.
CONCLUSIONS
In a large animal model, anthracycline exposure was associated with aortic fibrosis. In patients with lymphoma, anthracycline exposure was not associated with aortic inflammation.Further research is required to elucidate the mechanisms of anthracycline-related vascular harm.
PubMed: 38895275
DOI: 10.1101/2024.05.30.596741 -
Journal of Inflammation Research 2024Periductal mastitis (PDM) is a chronic inflammatory lesion of the breast with an unknown etiology, and it is difficult for clinicians to differentiate it from...
PURPOSE
Periductal mastitis (PDM) is a chronic inflammatory lesion of the breast with an unknown etiology, and it is difficult for clinicians to differentiate it from granulomatous lobular mastitis (GLM), although they have different treatment strategies and prognosis. This study aimed to investigate the differences in their clinicopathologic features to inform treatment strategies.
PATIENTS AND METHODS
Between 2011 and 2020, 121 patients diagnosed with PDM and 57 patients with GLM were retrospective analysis. Patient data were extracted on demographics, clinical presentation, pathologic characteristics, treatments and clinical response. Histopathological evaluations were performed on core needle biopsy specimens. Immunohistochemical stains using antibodies against CD3, CD4, CD8, CD20, and CD138 was performed to define immune cell infiltration.
RESULTS
PDM patients had a higher median age compared to GLM patients (38 vs 32, p<0.001). PDM was primarily located in the areolar area, while GLM predominantly affected the peripheral quadrant of the breast (56.20% vs 75.44%, p<0.001). Histopathologically, more ductal dilatation (90.08% vs 3.51%, p<0.001), ductal wall thickening (47.93% vs 1.75%, p<0.001), and ductal rupture (44.63% vs 5.26%, p<0.001) were observed in PDM. GLM presented with significantly more granuloma (94.74% vs 10.74%, p<0.001), microabscess (68.42% vs 28.93%, p<0.001), and lipid vacuole (40.35% vs 8.26%, p<0.001) formation than PDM. Immunohistochemical analysis revealed a significant presence of CD20+ B lymphocytes in PDM and a higher prevalence of CD8+ T lymphocytes in GLM, indicating differing immune responses. Treatment outcomes varied, with PDM patients responding well to surgery and anti-mycobacterial therapy, while GLM patients showed favorable responses to steroid therapy.
CONCLUSION
PDM is a specific entity with a similar clinical presentation but distinct histopathological features and immune profiles to GLM. Further research is needed to elucidate the pathogenesis and optimize therapeutic approaches for these breast inflammatory conditions.
PubMed: 38895142
DOI: 10.2147/JIR.S464585 -
International Journal of Molecular... Jun 2024Sprouty-related enabled/vasodilator-stimulated phosphoprotein homology 1 domain containing 2 (SPRED2) is an inhibitor of the mitogen-activated protein kinase...
Sprouty-related enabled/vasodilator-stimulated phosphoprotein homology 1 domain containing 2 (SPRED2) is an inhibitor of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway and has been shown to promote autophagy in several cancers. Here, we aimed to determine whether SPRED2 plays a role in autophagy in hepatocellular carcinoma (HCC) cells. The Cancer Genome Atlas (TCGA) Liver Cancer Database showed a negative association between the level of SPRED2 and p62, a ubiquitin-binding scaffold protein that accumulates when autophagy is inhibited. Immunohistochemically, accumulation of p62 was detected in human HCC tissues with low SPRED2 expression. Overexpression of SPRED2 in HCC cells increased the number of autophagosomes and autophagic vacuoles containing damaged mitochondria, decreased p62 levels, and increased levels of light-chain-3 (LC3)-II, an autophagy marker. In contrast, SPRED2 deficiency increased p62 levels and decreased LC3-II levels. SPRED2 expression levels were negatively correlated with translocase of outer mitochondrial membrane 20 (TOM20) expression levels, suggesting its role in mitophagy. Mechanistically, SPRED2 overexpression reduced ERK activation followed by the mechanistic or mammalian target of rapamycin complex 1 (mTORC1)-mediated signaling pathway, and SPRED2 deficiency showed the opposite pattern. Finally, hepatic autophagy was impaired in the liver of SPRED2-deficient mice with hepatic lipid droplet accumulation in response to starvation. These results indicate that SPRED2 is a critical regulator of autophagy not only in HCC cells, but also in hepatocytes, and thus the manipulation of this process may provide new insights into liver pathology.
Topics: Humans; Carcinoma, Hepatocellular; Liver Neoplasms; Autophagy; Hepatocytes; Animals; Mice; Cell Line, Tumor; Mechanistic Target of Rapamycin Complex 1; MAP Kinase Signaling System; Mitophagy; Repressor Proteins
PubMed: 38892460
DOI: 10.3390/ijms25116269 -
International Journal of Molecular... Jun 2024This study demonstrated the anticancer efficacy of chalcones with indole moiety (MIPP, MOMIPP) in fibrosarcoma cells for the first time. The results showed that MIPP and...
This study demonstrated the anticancer efficacy of chalcones with indole moiety (MIPP, MOMIPP) in fibrosarcoma cells for the first time. The results showed that MIPP and MOMIPP reduced the viability of HT-1080 cells in a concentration-dependent manner. MOMIPP was more active than MIPP in HT-1080 cells, showing lower IC values (3.67 vs. 29.90 μM). Both compounds at a concentration of 1 μM induced apoptosis in HT-1080 cells, causing death strictly related to caspase activation, as cell viability was restored when the caspase inhibitor Z-VAD was added. Reactive oxygen species production was approximately 3-fold higher than in control cells, and cotreatment with the inhibitor of mitochondrial ATPase oligomycin diminished this effect. Such effects were also reflected in mitochondrial dysfunction, including decreased membrane potential. Interestingly, the compounds that were studied caused massive vacuolization in HT-1080 cells. Immunocytochemical staining and TEM analysis showed that HT-1080 cells exhibited increased expression of the LC3-II protein and the presence of autophagosomes with a double membrane, respectively. Both compounds induced apoptosis, highlighting a promising link between autophagy and apoptosis. This connection could be a new target for therapeutic strategies to overcome chemoresistance, which is a significant cause of treatment failure and tumour recurrence in fibrosarcoma following traditional chemotherapy.
Topics: Humans; Apoptosis; Fibrosarcoma; Autophagy; Indoles; Cell Line, Tumor; Reactive Oxygen Species; Chalcones; Membrane Potential, Mitochondrial; Cell Survival; Antineoplastic Agents; Mitochondria
PubMed: 38892288
DOI: 10.3390/ijms25116100 -
International Journal of Molecular... May 2024Podophyllotoxin (PPT) is an active pharmaceutical ingredient (API) with established antitumor potential. However, due to its systemic toxicity, its use is restricted to...
Cellular Distribution and Ultrastructural Changes in HaCaT Cells, Induced by Podophyllotoxin and Its Novel Fluorescent Derivative, Supported by the Molecular Docking Studies.
Podophyllotoxin (PPT) is an active pharmaceutical ingredient (API) with established antitumor potential. However, due to its systemic toxicity, its use is restricted to topical treatment of anogenital warts. Less toxic PPT derivatives (e.g., etoposide and teniposide) are used intravenously as anticancer agents. PPT has been exploited as a scaffold of new potential therapeutic agents; however, fewer studies have been conducted on the parent molecule than on its derivatives. We have undertaken a study of ultrastructural changes induced by PPT on HaCaT keratinocytes. We have also tracked the intracellular localization of PPT using its fluorescent derivative (PPT-FL). Moreover, we performed molecular docking of both PPT and PPT-FL to compare their affinity to various binding sites of tubulin. Using the Presto blue viability assay, we established working concentrations of PPT in HaCaT cells. Subsequently, we have used selected concentrations to determine PPT effects at the ultrastructural level. Dynamics of PPT distribution by confocal microscopy was performed using PPT-FL. Molecular docking calculations were conducted using Glide. PPT induces a time-dependent cytotoxic effect on HaCaT cells. Within 24 h, we observed the elongation of cytoplasmic processes, formation of cytoplasmic vacuoles, progressive ER stress, and shortening of the mitochondrial long axis. After 48 h, we noticed disintegration of the cell membrane, progressive vacuolization, apoptotic/necrotic vesicles, and a change in the cell nucleus's appearance. PPT-FL was detected within HaCaT cells after ~10 min of incubation and remained within cells in the following measurements. Molecular docking confirmed the formation of a stable complex between tubulin and both PPT and PPT-FL. However, it was formed at different binding sites. PPT is highly toxic to normal human keratinocytes, even at low concentrations. It promptly enters the cells, probably via endocytosis. At lower concentrations, PPT causes disruptions in both ER and mitochondria, while at higher concentrations, it leads to massive vacuolization with subsequent cell death. The novel derivative of PPT, PPT-FL, forms a stable complex with tubulin, and therefore, it is a useful tracker of intracellular PPT binding and trafficking.
Topics: Humans; Molecular Docking Simulation; Podophyllotoxin; HaCaT Cells; Tubulin; Keratinocytes; Cell Survival; Mitochondria; Fluorescent Dyes; Binding Sites; Endoplasmic Reticulum Stress
PubMed: 38892135
DOI: 10.3390/ijms25115948 -
Plants (Basel, Switzerland) May 2024Parkinson's disease (PD) is a leading neurodegenerative disorder affecting 1-3 percent of the elderly population. Oxidative stress is the primary factor for the...
Parkinson's disease (PD) is a leading neurodegenerative disorder affecting 1-3 percent of the elderly population. Oxidative stress is the primary factor for the neurodegeneration of Substantia Nigra (SN). The current study aims to assess the seed extracts of (MO) on rotenone-mediated motor function impairments in a PD mouse model. For this purpose, two different seed extracts of MO were prepared, including aqueous MO (AqMO) and ethanolic MO (EthMO). Male Swiss albino mice were grouped into five groups. Mice received 2.5 mg/kg rotenone for 21 consecutive days, and control mice received the vehicle. Extract-treated mice received 200 mg/kg AqMO and EthMO separately, orally and daily for 28 days. Sinemet-treated mice received 20 mg/kg, oral dose, as a positive group. The motor function performance was evaluated using standard neurobehavioral tests. The antioxidant potentials of MO seed extracts were estimated by lipid peroxidation (LPO), reduced glutathione (GSH), glutathione-s-transferase (GST) and catalase (CAT) activities in mice brain homogenates. The PD mice brain SN sections were investigated for neurodegeneration. MO seed extract-treated mice showed a significant reduction in motor dysfunction compared to rotenone-treated mice as assessed through the open field, beam walk, pole climb-down, tail suspension, stride length and stepping tests. Increased antioxidant capacities of the PD mice brains of MO extract-administered groups were observed compared to the control. A histological study showed reduced signs of neurodegeneration, vacuolation around multipolar cells and cytoplasmic shrinkage in MO extract-treated mice SN brain sections. Collectively, MO seed extracts protected the animals from locomotor deficits induced by rotenone, possibly through antioxidant means, and seem to have potential applications in neurodegenerative diseases.
PubMed: 38891288
DOI: 10.3390/plants13111479 -
Cells May 2024PIKfyve is an endosomal lipid kinase that synthesizes phosphatidylinositol 3,5-biphosphate from phosphatidylinositol 3-phsphate. Inhibition of PIKfyve activity leads to...
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.
Topics: Animals; Zebrafish; TOR Serine-Threonine Kinases; Signal Transduction; Phosphatidylinositol 3-Kinases; Lysosomal Storage Diseases; Lysosomes; Humans; Phosphoinositide-3 Kinase Inhibitors; Zebrafish Proteins
PubMed: 38891085
DOI: 10.3390/cells13110953